Microorganisms and processes for producing terephthalic acid and its salts

ABSTRACT

The invention provides non-naturally occurring microbial organisms having a (2-hydroxy-3-methyl-4-oxobutoxy) phosphonate (2H3M40P) pathway, p-toluate pathway, and/or terephthalate pathway. The invention additionally provides methods of using such organisms to produce 2H3M40P, p-toluate or terephthalate. Also provided herein are processes for isolating bio-based aromatic carboxylic acid, in particular, p-toluic acid or terephthalic acid, from a culture medium, wherein the processes involve contacting the culture medium with sufficient carbon dioxide (C02) to lower the pH of the culture medium to produce a precipitate comprised of the aromatic carboxylic acid.

This application claims the benefit of priority of U.S. Provisionalapplication Ser. No. 61/589,081, filed Jan. 20, 2012, and U.S.Provisional application Ser. No. 61/598,743, filed Feb. 14, 2012, theentire contents of which are incorporated herein by reference.

TECHNICAL FIELD

The present invention relates generally to biosynthetic processes, andmore specifically to organisms having(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate (2H3M4OP), p-toluate orterephthalate biosynthetic capability. Also provided herein areprocesses for isolating a bio-based aromatic carboxylic acid, forexample, p-toluic acid or terephthalic acid, from a culture medium,wherein the processes involve lowering the pH of the culture medium toproduce a precipitate comprised of the aromatic carboxylic acid.

BACKGROUND OF THE INVENTION

Terephthalate (also known as terephthalic acid and PTA) is the immediateprecursor of polyethylene terephthalate (PET), used to make clothing,resins, plastic bottles and even as a poultry feed additive. Nearly allPTA is produced from para-xylene by oxidation in air in a process knownas the Mid Century Process. This oxidation is conducted at hightemperature in an acetic acid solvent with a catalyst composed of cobaltand/or manganese salts. Para-xylene is derived from petrochemicalsources and is formed by high severity catalytic reforming of naphtha.Xylene is also obtained from the pyrolysis gasoline stream in a naphthasteam cracker and by toluene disproportion.

Cost-effective methods for generating renewable PTA have not yet beendeveloped to date. PTA, toluene and other aromatic precursors arenaturally degraded by some bacteria. However, these degradation pathwaystypically involve monooxygenases that operate irreversibly in thedegradative direction. Hence, biosynthetic pathways for PTA are severelylimited by the properties of known enzymes to date.

A promising precursor for PTA is p-toluate, also known asp-methylbenzoate. P-Toluate is an intermediate in some industrialprocesses for the oxidation of p-xylene to PTA. It is also anintermediate for polymer stabilizers, pesticides, light sensitivecompounds, animal feed supplements and other organic chemicals. Onlyslightly soluble in aqueous solution, p-toluate is a solid atphysiological temperatures, with a melting point of 275° C. Microbialcatalysts for synthesizing this compound from sugar feedstocks have notbeen described to date.

Petrochemical based chemical syntheses for making terephthalic acid areknown (see, e.g., U.S. Pat. Nos. 2,905,709; 3,023,234; 3,042,717;3,043,846; 3,064,041; 3,096,366; and 6,441,225). Alternativetechnologies for the production of terephthalic acid have been madefeasible with the advent of molecular recombinant technologies used tomodify biosynthetic pathways in microbial organisms. For example,microbial organisms have been described which produce precursors usefulfor the synthesis of bio-based terephthalic acid. Exemplary indirectsemi-synthetic routes of producing bio-based terephthalic acid aredescribed in U.S. Patent Publication No. 2011/0124911 A1. Directbiosynthetic routes, wherein terephthalate is itself biosynthesized inmicrobial organisms have been described in U.S. Patent Publication No.2011/0207185 A1. Even with advantages that bio-based production ofterephthalic acid offers, improvements and/or additional processes aresought, for example, to improve recovery of terephthalic acid in termsof yields and purity, and to increase efficiency and scalability of theprocesses including, for instance, reducing the number of manufacturingsteps, lowering energy usage, recapturing and recycling materials andreducing environmental discharges.

Thus, there exists a need for alternative methods for effectivelyproducing and isolating commercial quantities of compounds such asp-toluate or terephthalate. The present invention satisfies this needand provides related advantages as well.

SUMMARY OF INVENTION

The invention provides non-naturally occurring microbial organismshaving a 2H3M4OP pathway, p-toluate pathway, and/or terephthalatepathway. The invention additionally provides methods of using suchorganisms to produce 2H3M4OP, p-toluate or terephthalate.

The invention also provides a process for isolating a bio-based aromaticcarboxylic acid from a culture medium. In certain embodiments, theprocess comprises the steps of: (a) culturing a non-naturally occurringmicrobial organism in a culture medium to produce an aromaticcarboxylate anion at a pH sufficient to maintain the aromaticcarboxylate anion in soluble form; (b) lowering the pH of the culturemedium to produce an aromatic carboxylic acid precipitate from thearomatic carboxylate anion. In certain embodiments, lowering the pH ofthe culture medium comprises contacting the culture medium with carbondioxide (CO₂). In certain embodiments, the culture medium issubstantially depleted of the aromatic carboxylate anion. In certainembodiments, the process further comprises separating the culture mediumfrom non-soluble materials, for example, cells, cell debris, cellulosicmaterial, feed stock, etc., prior to lowering the pH. In certainembodiments, the process further comprises separating the aromaticcarboxylic acid from the culture medium, for example, by centrifugationor membrane filtration, etc.

In certain embodiments of the isolation process, the aromatic carboxylicacid is p-toluic acid. In certain embodiments, the aromatic carboxylicacid is terephthalic acid.

In certain embodiments of the isolation process, a counter ion to thearomatic carboxylate anion in the culture medium is an ammonium, sodiumor potassium cation. In certain embodiments, the counter ion is anammonium cation.

In certain embodiments of the isolation process, the pH sufficient tomaintain the aromatic carboxylate anion in soluble form is between about5.0-9.0 pH units. In some embodiments, the pH is about 6.0 to about 8.0pH units or about 6.2 to about 7.8 pH units. In certain embodiments, thepH sufficient to maintain the aromatic carboxylate anion in soluble formis about 7.0 pH units. In certain embodiments, base is added to theculture medium in the culturing step to maintain the aromaticcarboxylate anion in soluble form. In certain embodiments, the base isammonia.

In certain embodiments of the isolation process, when lowering the pH ofthe culture medium, the pH is lowered to less than about 5.0 pH units,less than about 4.5 pH units, less than about 4.0 pH units, less thanabout 3.5 pH units, less than about 3.0 pH units, less than about 2.5 pHunits, less than about 2.0 pH units, less than about 1.5 pH units, orless than about 1.0 pH units. In certain embodiments, the pH of theculture medium following the first separation step is lowered to lessthan about 3.0 pH units. In certain embodiments, the pH is lowered inthe culture medium following separation of culture medium fromnon-soluble materials such as cells, cell debris, feedstock, and thelike, which, for example, can be present when culturing a non-naturallyoccurring microbial organism.

In certain embodiments of the isolation process, wherein the culturemedium is contacted with CO₂, the CO₂ used in the contacting step isgaseous. In certain embodiments, the gaseous CO₂ is pure CO₂ gas. Incertain embodiments, the gaseous CO₂ is in a mixture with one or moreadditional gases. In certain embodiments, the additional gas is nitrogengas. In certain embodiments, CO₂ generated from the culturing step canbe used to lower the pH of the culture medium.

In certain embodiments of the isolation process, the culture medium iscontacted with CO₂ in the range of 0.1 to 30 atm. In certainembodiments, the culture medium is stirred at temperatures between 0° C.and 80° C. for up to 24 hours during this contacting step.

In certain embodiments of the isolation process, when present, thesecond separation step comprises filtering and recovering of thearomatic carboxylic acid from the culture medium.

In certain embodiments of the isolation process, the process furthercomprises purifying the separated aromatic carboxylic acid. In certainembodiments, the purification step comprises crystallizing the aromaticcarboxylic acid.

In certain embodiments of the isolation process, the non-naturallyoccurring microbial organism produces p-toluate and the aromaticcarboxylic acid is p-toluic acid.

In certain embodiments of the isolation process, the non-naturallyoccurring microbial organism produces terephthalate and the aromaticcarboxylic acid is terephthalic acid.

In certain embodiments of the isolation process, the non-naturallyoccurring microbial organism has a 2H3M4OP pathway, p-toluate pathway,and/or terephthalate pathway.

In certain embodiments of the isolation process, the non-naturallyoccurring microbial organism produces muconate, and the process furthercomprises contacting muconate with acetylene to form a cyclohexadieneadduct, and oxidizing the cyclohexadiene adduct to form the aromaticcarboxylate anion. In certain embodiments, wherein the non-naturallyoccurring microbial organism produces muconate, the process forisolating a bio-based aromatic carboxylic acid from a culture mediumcomprises the steps of: (a) culturing a non-naturally occurringmicrobial organism in a culture medium to produce muconate at a pHsufficient to maintain muconate in soluble form; (b) contacting muconatewith acetylene to form a cyclohexadiene adduct; (c) oxidizing thecyclohexadiene adduct to form the aromatic carboxylate anion; and (d)contacting the culture medium with sufficient carbon dioxide (CO₂) tolower the pH of the culture medium to produce an aromatic carboxylicacid precipitate, wherein the culture medium is substantially depletedof the aromatic carboxylate anion. In certain embodiments, the processfurther comprises separating the culture medium from non-solublematerials, for example, cells, cell debris, cellulosic material, feedstock, etc., prior to lowering the pH. In certain embodiments, theprocess further comprises separating the aromatic carboxylic acid fromthe culture medium, for example, by centrifugation or membranefiltration, etc. In certain embodiments, the aromatic carboxylic acid isterephthalic acid.

In another aspect of the isolation process, an isolated aromaticcarboxylic acid produced by the processes disclosed herein is provided.In certain embodiments, isolated bio-based p-toluic acid is produced. Incertain embodiments, isolated bio-based terephthalic acid is produced.

Terephthalate produced by a microorganism or isolation process of theinvention can be used as a precursor for production of a polymer,including polyethylene terephthalate (PET), polybutyl terephthalate(PBT) or polytrimethylene terephthalate (PTT). PET can be produced byreacting ethylene glycol with dimethyl terephthalate of the invention bytransesterification or by reacting ethylene glycol with terephthalate ofthe invention by esterification. PBT can be produced by reacting1,4-butanediol with terephthalate of the invention. PTT can be producedby reacting 1,3-propanediol with terephthalate of the invention.Accordingly, in certain embodiments, the invention provides PET, PBT orPTT comprising, obtained by or manufactured using the terephthalateproduced by a microorganism of the invention or isolated by a processdescribed herein. Furthermore, PET can be used to manufacture bulkmaterials such as, for example, chips (e.g. PET bottle chips), resinsand fibers, which in turn can be used to make cloth, clothing andplastic bottles, or even used as a poultry feed additive. PBT can beused to manufacture several products, such as, for example, moldedarticles, injection-molded products, injection-molded parts, such as anautomotive part, extrusion resins, electrical parts or casings. PTT canalso be used to manufacture several products, including, for example,fibers, cloth, carpets or bottles.

In certain embodiments, the invention provides a process for obtainingPET by reacting ethylene glycol with dimethyl terephthalate, wherein thedimethyl terephthalate is produced from terephthalate produced by amicroorganism of the invention or isolated by a process describedherein. In another aspect, the invention provides a process forobtaining PET by reacting ethylene glycol with terephthalic acid,wherein the terephthalic acid is produced by a microorganism of theinvention or isolated by a process described herein.

In certain embodiments, the invention provides a process for obtainingPBT by reacting 1,4-butanediol with terephthalate produced by amicroorganism of the invention or isolated by a process describedherein.

In certain embodiments, the invention provides a process for obtainingPTT by reacting 1,3-propanediol with terephthalate produced by amicroorganism of the invention or isolated by a process describedherein, or reacting 1,3-propanediol with dimethyl terephthalate, whereinthe dimethyl terephthalate is produced from the terephthalate producedby a microorganism of the invention or isolated by a process describedherein.

In certain embodiments, the invention provides a polyester fiber, apolyester cloth or a polyester carpet comprising, obtained by ormanufactured using PET or PTT, wherein the PET or PTT comprises, wasobtained by or was manufactured using the terephthalate produced by amicroorganism of the invention or isolated by a process describedherein.

In certain embodiments, the invention provides a chip comprising,obtained by or manufactured using PET or PTT, wherein the PET or PTTcomprises, was obtained by or was manufactured using the terephthalateproduced by a microorganism of the invention or isolated by a processdescribed herein. In another aspect, the invention provides a PET or PTTbottle comprising, obtained by or manufactured using the chips describedherein.

In certain embodiments, the invention provides a packaging containercomprising, obtained by or manufactured using PET, wherein the PETcomprises, was obtained by or was manufactured using the terephthalateproduced by a microorganism of the invention or a process describedherein.

In certain embodiments, the invention provides a film comprising,obtained by or manufactured using PET, wherein the PET comprises, wasobtained by or was manufactured using the terephthalate produced by amicroorganism of the invention or a process described herein.

In certain embodiments, the invention provides a molded articlecomprising, obtained by or manufactured using PET, PBT or PTT, whereinthe PET, PBT or PTT comprises, was obtained by or was manufactured usingthe terephthalate produced by a microorganism of the invention or aprocess described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic depiction of an exemplary pathway fromerythrose-4-phosphate to (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate(“2H3M4OP”). In steps A-F, the enzymes can be: A. erythrose-4-phosphatedehydrogenase; B. 4-phosphoerythronate dehydrogenase; C.2-acetyl-2,3-dihydroxy-4-phosphobutanoate synthase; D.2-acetyl-2,3-dihydroxy-4-phosphobutanoate reductoisomerase; E.2,3,4-trihydroxy-3-methyl-5-phosphopentanoate dehydratase; and F.4-hydroxy-3-methyl-2-oxo-5-phosphopentanoate decarboxylase.

FIG. 2 shows a schematic depiction of exemplary pathways to 2H3M4OP from4,5-dihydroxy-2-oxopentanoate. In steps A-E, the enzymes can be: A.4,5-dihydroxy-2-oxopentanoate methyltransferase; B.4,5-dihydroxy-3-methyl-2-oxopentanoate kinase; C.4-hydroxy-3-methyl-2-oxo-5-phosphopentanoate decarboxylase; D.4,5-dihydroxy-2-oxopentanoate kinase; and E.4-hydroxy-2-oxo-5-phosphopentanoate methyltransferase. 2H3M4OP is(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate.

FIG. 3 shows a schematic depiction of an exemplary pathway to 2H3M4OPfrom glyceraldehyde-3-phosphate and pyruvate. G3P isglyceraldehyde-3-phosphate, DXP is 1-deoxy-D-xylulose-5-phosphate, 2ME4Pis C-methyl-D-erythritol-4-phosphate and 2H3M4OP is(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate. In steps A-C, the enzymescan be: A. DXP synthase; B. DXP reductoisomerase; and C. 2ME4Pdehydratase.

FIG. 4 shows a schematic depiction of an exemplary alternate shikimatepathway to p-toluate. In steps A-H, the enzymes can be: A.2-dehydro-3-deoxyphosphoheptonate synthase; B. 3-dehydroquinatesynthase; C. 3-dehydroquinate dehydratase; D. shikimate dehydrogenase;E. Shikimate kinase; F. 3-phosphoshikimate-2-carboxyvinyltransferase; G.chorismate synthase; and H. chorismate lyase. Compounds are: (1)(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate; (2)2,4-dihydroxy-5-methyl-6-[(phosphonooxy)methyl]oxane-2-carboxylate; (3)1,3-dihydroxy-4-methyl-5-oxocyclohexane-1-carboxylate; (4)5-hydroxy-4-methyl-3-oxocyclohex-1-ene-1-carboxylate; (5)3,5-dihydroxy-4-methylcyclohex-1-ene-1-carboxylate; (6)5-hydroxy-4-methyl-3-(phosphonooxy)cyclohex-1-ene-1-carboxylate; (7)5-[(1-carboxyeth-1-en-1-yl)oxy]-4-methyl-3-(phosphonooxy)cyclohex-1-ene-1-carboxylate;(8)3-[(1-carboxyeth-1-en-1-yl)oxy]-4-methylcyclohexa-1,5-diene-1-carboxylate;and (9) p-toluate.

FIG. 5 shows a schematic depiction of an exemplary pathway forconversion of p-toluate to terephthalic acid (PTA). Reactions A, B and Care catalyzed by p-toluate methyl-monooxygenase reductase,4-carboxybenzyl alcohol dehydrogenase and 4-carboxybenzyl aldehydedehydrogenase, respectively. The compounds shown are (1) p-toluic acid;(2) 4-carboxybenzyl alcohol; (3) 4-carboxybenzaldehyde and (4)terephthalic acid.

FIG. 6 depicts an exemplary process for preparing bio-based terephthalicacid.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to, at least in part, the design andproduction of cells and organisms having biosynthetic productioncapabilities for 2H3M4OP, p-toluate or terephthalate. The resultsdescribed herein indicate that metabolic pathways can be designed andrecombinantly engineered to achieve the biosynthesis of 2H3M4OP,p-toluate or terephthalate in Escherichia coli and other cells ororganisms. Biosynthetic production of 2H3M4OP, p-toluate orterephthalate can be confirmed by construction of strains having thedesigned metabolic genotype. These metabolically engineered cells ororganisms also can be subjected to adaptive evolution to further augment2H3M4OP, p-toluate or terephthalate biosynthesis, including underconditions approaching theoretical maximum growth.

The shikimate biosynthesis pathway in E. coli convertserythrose-4-phosphate to chorismate, an important intermediate thatleads to the biosynthesis of many essential metabolites including4-hydroxybenzoate. 4-Hydroxybenzoate is structurally similar top-toluate, an industrial precursor of terephthalic acid. As disclosedherein, shikimate pathway enzymes can be utilized to accept thealternate substrate, 2H3M4OP and transform it to p-toluate. In addition,various pathway enzymes can be used to synthesize the 2H3M4OP precursorfrom erythrose-4-phosphate using enzymes for P5C biosynthesis andenzymes analogous to the isoleucine biosynthesis pathway, oralternatively from 4,5-dihydroxy-2-oxopentanoate using a methyltransferase, a kinase and a decarboxylase. Synthesis of the 2H3M4OPprecursor from glyceraldehydes-3-phosphate and pyruvate can also be doneusing enzymes from the non-mevalonate pathway for isoprenoidbiosynthesis.

Disclosed herein are strategies for engineering a microorganism toproduce renewable p-toluate or terephthalate (PTA) from carbohydratefeedstocks. The substrate 4,5-dihydroxy-2-oxopentanoate is naturallyderived from sugars such as arabinose and xylose. Additionally, thissubstrate can be formed enzymatically by condensation of pyruvate andglycolaldehyde by aldolase enzymes such as 2-dehydro-3-deoxypentonatealdolase, 2-dehydro-3-deoxyglucarate aldolase, or other enzymes in ECclass 4.1.2 or 4.1.3. The substrate erythrose-4-phosphate is anintermediate in the pentose phosphate pathway and the Calvin cycle.Additionally, erythrose-4-phosphate can serve as a precursor to thebiosynthesis of the aromatic amino acids tyrosine, phenylalanine andtryptophan. First, erythrose-4-phosphate can be converted to 2H3M4OP insix enzymatic steps (see Example I and FIG. 1). In one alternative,4,5-dihydroxy-2-oxopentanoate is converted to 2H3M4OP using one or bothof the pathways described in Example I and FIG. 2. In anotheralternative, glyceraldehyde-3-phosphate (G3P) and pyruvate are convertedto 2H3M4OP in three enzymatic steps (see Example II and FIG. 3). The2H3M4OP intermediate can be subsequently transformed to p-toluate byenzymes in the shikimate pathway (see Example III and FIG. 4). p-Toluatecan be further converted to PTA (terephthalate) by a microorganism (seeExample IV and FIG. 5).

The purification method of the invention, e.g. exemplified in FIG. 6,can also be applied to biosynthetic pathways for aromatic carboxylicacid and terephthalic acid production described in WIPO patentpublications WO/2009/120457A2 entitled “Bio-Based PolyethyleneTerephthalate Polymer And Method Of Making The Same”, WO/2011/094131A1entitled “Microorganisms And Methods For The Biosynthesis Of P-ToluateAnd Terephthalate”, and WO/2011/017560A1 entitled “Semi-SyntheticTerephthalic Acid Via Microorganisms That Produce Muconic Acid” and U.S.Pat. No. 6,461,840 entitled “Terephthalic acid producing proteobacteria”and U.S. Pat. No. 6,187,569 entitled “Microbial production ofterephthalic acid and isophthalic acid.”

The maximum theoretical PTA yield from glucose via the proposederythrose-4-phosphate pathway in FIG. 1, in conjunction with thepathways from 2H3M4OP to PTA in FIGS. 4 and 5, is 0.6 moles of PTA permole of glucose utilized (0.55 g/g). Increasing product yields to 0.61mol/mol (0.56 g/g) is possible if cells are capable of fixing CO₂through pathways such as the reductive TCA cycle or the Wood-Ljungdahlpathway.

The maximum theoretical PTA yield from xylose via the4,5-dihydroxy-2-oxopentanoate pathway of FIG. 2 is 0.46 moles PTA permole xylose utilized (0.51 g/g).

The conversion of G3P to p-toluate requires one ATP, two reducingequivalents (NAD(P)H), and two molecules of phosphoenolpyruvate,according to net reaction below.

G3P+2PEP+ATP+2NAD(P)H+2H⁺ p-Toluate+4Pi+ADP+2NAD(P)⁺+CO₂+H₂O

One equivalent of CO₂ is generated in this net reaction.

An additional ATP is required to synthesize G3P from glucose. Themaximum theoretical p-toluate yield is 0.67 mol/mol (0.51 g/g) fromglucose minus carbon required for energy. Under the assumption that 2ATPs are consumed per p-toluate molecule synthesized, the predictedp-toluate yield from glucose is 0.62 mol/mol (0.46 g/g) p-toluate.

If p-toluate is further converted to PTA by enzymes as described inExample IV, the predicted PTA yield from glucose is 0.64 mol/mol (0.58g/g). In this case, the oxidation of p-toluate to PTA generates anadditional net reducing equivalent according to the net reaction:

p-toluate+O₂+NAD⁺PTA+NADH+2H⁺

Enzyme candidates for catalyzing each step of the proposed pathways aredescribed in the following sections.

As used herein, the term “non-naturally occurring” when used inreference to a microbial organism or microorganism of the invention isintended to mean that the microbial organism has at least one geneticalteration not normally found in a naturally occurring strain of thereferenced species, including wild-type strains of the referencedspecies. Genetic alterations include, for example, modificationsintroducing expressible nucleic acids encoding metabolic polypeptides,other nucleic acid additions, nucleic acid deletions and/or otherfunctional disruption of the microbial organism's genetic material. Suchmodifications include, for example, coding regions and functionalfragments thereof, for heterologous, homologous or both heterologous andhomologous polypeptides for the referenced species. Additionalmodifications include, for example, non-coding regulatory regions inwhich the modifications alter expression of a gene or operon. Exemplarymetabolic polypeptides include enzymes or proteins within a muconate,2H3M4OP, p-toluate and/or terephthalate biosynthetic pathway.

A metabolic modification refers to a biochemical reaction that isaltered from its naturally occurring state. Therefore, non-naturallyoccurring microorganisms can have genetic modifications to nucleic acidsencoding metabolic polypeptides, or functional fragments thereof.Exemplary metabolic modifications are disclosed herein.

As used herein, the term “isolated” when used in reference to amicrobial organism is intended to mean an organism that is substantiallyfree of at least one component as the referenced microbial organism isfound in nature. The term includes a microbial organism that is removedfrom some or all components as it is found in its natural environment.The term also includes a microbial organism that is removed from some orall components as the microbial organism is found in non-naturallyoccurring environments. Therefore, an isolated microbial organism ispartly or completely separated from other substances as it is found innature or as it is grown, stored or subsisted in non-naturally occurringenvironments. Specific examples of isolated microbial organisms includepartially pure microbes, substantially pure microbes and microbescultured in a medium that is non-naturally occurring.

As used herein, the terms “microbial,” “microbial organism” or“microorganism” are intended to mean any organism that exists as amicroscopic cell that is included within the domains of archaea,bacteria or eukarya. Therefore, the term is intended to encompassprokaryotic or eukaryotic cells or organisms having a microscopic sizeand includes bacteria, archaea and eubacteria of all species as well aseukaryotic microorganisms such as yeast and fungi. The term alsoincludes cell cultures of any species that can be cultured for theproduction of a biochemical.

As used herein, the term “(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate,”abbreviated herein as 2H3M4OP, has the chemical formula as shown inFIG. 1. Such a compound can also be described as 3-hydroxy-2-methylbutanal-4-phosphate.

As used herein, the term “p-toluate,” having the molecular formulaC₈H₇O₂ ⁻ (see FIG. 4, compound 9) (IUPAC name 4-methylbenzoate) is theionized form of p-toluic acid, and it is understood that p-toluate andp-toluic acid can be used interchangeably throughout to refer to thecompound in any of its neutral or ionized forms, including any saltforms thereof. It is understood by those skilled understand that thespecific form will depend on the pH.

As used herein, the term “terephthalate,” having the molecular formulaC₈H₄O₄ ⁻² (see FIG. 5, compound 4)(IUPAC name terephthalate) is theionized form or dianionic form of terephthalic acid, also referred to asp-phthalic acid or PTA, depicted below.

Those skilled in the art will understand that occurrences ofterephthalate and terephthalic acid in solution will depend on pH,moreover the terms “terephthalate” and “terephthalic acid,” unlessotherwise indicated in the context that they are used, can be usedinterchangeably throughout to refer to the compound in any of itsneutral or ionized forms, including any salt forms thereof and are notintended to be limiting to one specific form, e.g., its neutral or acidform, or ionized forms, including any salt forms thereof. Asterephthalic acid is a dicarboxylic acid, it can exist in a partiallyprotonated monoacid form or a fully protonated diacid form, dependent onthe pH. Unless otherwise specified, as used herein “terephthalic acid”will refer to the fully protonated diacid form.

As used herein, the term “bio-based” means derived from or synthesizedby a biological organism and can be considered a renewable resourcesince it can be generated by a biological organism. Such a biologicalorganism, in particular the microbial organisms of the inventiondisclosed herein, can utilize feedstock or biomass, such as sugars,glycerol or carbohydrates obtained from an agricultural, plant,bacterial, or animal source. Alternatively, the biological organism canutilize atmospheric carbon, carbon dioxide, formate, methane, methanol,carbon in the form of syngas or a carbon source generated fromelectrochemical conversion of carbon dioxide.

As used herein, the term “bio-derived” means a product as describedherein that is composed, in whole or in part, of a bio-based compound ofthe invention. A bio-derived or bio-based product is in contrast to apetroleum derived product, wherein such a product is derived from orsynthesized from petroleum or a petrochemical feedstock.

As used herein, the term “muconate” is an ionized or anionic form ofmuconic acid depicted below.

Those skilled in the art will understand that occurrences of muconateand muconic acid in solution will depend on pH, moreover the terms“muconate” and “muconic acid,” unless otherwise indicated in the contextthat they are used, are not intended to be limiting to one specificform, e.g., its neutral or acid form, or ionized forms, including anysalt forms thereof.

As used herein, the term “aromatic carboxylic acid” refers to a compoundthat contains one or more carboxylate (COOH) groups, bonded to anaromatic ring. Examples of aromatic carboxylic acids include benzoicacid, salicylic acid, gallic acid, o-toluic acid, m-toluic acid,p-toluic acid, phthalic acid, isopthalic acid, and terephthalic acid, asdepicted below.

Those skilled in the art will understand that certain aromaticcarboxylic acids are mono carboxylic acids (monoacids), such as p-toluicacid, whereas certain aromatic carboxylic acids are di carboxylic acids(diacids), such as terephthalic acid.

As used herein, the term “aromatic carboxylate anion” refers to theconjugate base of the aromatic carboxylic acid. Those skilled in the artwill understand that when a carboxyl group is deprotonated, thecarboxylate anion is formed. Those skilled in the art will furtherunderstand that the specific form of the aromatic carboxylic acid (i.e.,whether protonated as an acid, or deprotonated as an anion) will dependon the pH.

As used herein, the term “p-toluate” is the ionized or anionic form ofp-toluic acid, as depicted below.

Those skilled in the art will understand that occurrences of p-toluateand p-toluic acid in solution will depend on pH, moreover the terms“p-toluate” and “p-toluic acid,” unless otherwise indicated in thecontext that they are used, are not intended to be limiting to onespecific form, e.g., its neutral or acid form, or ionized forms,including any salt forms thereof.

As used herein, the terms “about” or “approximately” means an acceptableerror for a particular value as determined by those of skill in the art,which depends in part on how the value is measured or determined. Incertain embodiments, the terms “about” or “approximately” means within1, 2, 3, or 4 standard deviations. In certain embodiments, the term“about” or “approximately” means within above or below 20%, 15%, 10%,9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.05% of a given value orrange.

As used herein, the term “substantially anaerobic” when used inreference to a culture or growth condition is intended to mean that theamount of oxygen is less than about 10% of saturation for dissolvedoxygen in liquid media. The term also is intended to include sealedchambers of liquid or solid medium maintained with an atmosphere of lessthan about 1% oxygen.

“Exogenous” as it is used herein is intended to mean that the referencedmolecule or the referenced activity is introduced into the hostmicrobial organism. The molecule can be introduced, for example, byintroduction of an encoding nucleic acid into the host genetic materialsuch as by integration into a host chromosome or as non-chromosomalgenetic material such as a plasmid. Therefore, the term as it is used inreference to expression of an encoding nucleic acid refers tointroduction of the encoding nucleic acid in an expressible form intothe microbial organism. When used in reference to a biosyntheticactivity, the term refers to an activity that is introduced into thehost reference organism. The source can be, for example, a homologous orheterologous encoding nucleic acid that expresses the referencedactivity following introduction into the host microbial organism.Therefore, the term “endogenous” refers to a referenced molecule oractivity that is present in the host. Similarly, the term when used inreference to expression of an encoding nucleic acid refers to expressionof an encoding nucleic acid contained within the microbial organism. Theterm “heterologous” refers to a molecule or activity derived from asource other than the referenced species whereas “homologous” refers toa molecule or activity derived from the host microbial organism.Accordingly, exogenous expression of an encoding nucleic acid of theinvention can utilize either or both a heterologous or homologousencoding nucleic acid.

It is understood that when more than one exogenous nucleic acid isincluded in a microbial organism that the more than one exogenousnucleic acids refers to the referenced encoding nucleic acid orbiosynthetic activity, as discussed above. It is further understood, asdisclosed herein, that such more than one exogenous nucleic acids can beintroduced into the host microbial organism on separate nucleic acidmolecules, on polycistronic nucleic acid molecules, or a combinationthereof, and still be considered as more than one exogenous nucleicacid. For example, as disclosed herein a microbial organism can beengineered to express two or more exogenous nucleic acids encoding adesired pathway enzyme or protein. In the case where two exogenousnucleic acids encoding a desired activity are introduced into a hostmicrobial organism, it is understood that the two exogenous nucleicacids can be introduced as a single nucleic acid, for example, on asingle plasmid, on separate plasmids, can be integrated into the hostchromosome at a single site or multiple sites, and still be consideredas two exogenous nucleic acids. Similarly, it is understood that morethan two exogenous nucleic acids can be introduced into a host organismin any desired combination, for example, on a single plasmid, onseparate plasmids, can be integrated into the host chromosome at asingle site or multiple sites, and still be considered as two or moreexogenous nucleic acids, for example three exogenous nucleic acids.Thus, the number of referenced exogenous nucleic acids or biosyntheticactivities refers to the number of encoding nucleic acids or the numberof biosynthetic activities, not the number of separate nucleic acidsintroduced into the host organism.

The non-naturally occurring microbial organisms of the invention cancontain stable genetic alterations, which refers to microorganisms thatcan be cultured for greater than five generations without loss of thealteration. Generally, stable genetic alterations include modificationsthat persist greater than 10 generations, particularly stablemodifications will persist more than about 25 generations, and moreparticularly, stable genetic modifications will be greater than 50generations, including indefinitely.

Those skilled in the art will understand that the genetic alterations,including metabolic modifications exemplified herein, are described withreference to a suitable host organism such as E. coli and theircorresponding metabolic reactions or a suitable source organism fordesired genetic material such as genes for a desired metabolic pathway.However, given the complete genome sequencing of a wide variety oforganisms and the high level of skill in the area of genomics, thoseskilled in the art will readily be able to apply the teachings andguidance provided herein to essentially all other organisms. Forexample, the E. coli metabolic alterations exemplified herein canreadily be applied to other species by incorporating the same oranalogous encoding nucleic acid from species other than the referencedspecies. Such genetic alterations include, for example, geneticalterations of species homologs, in general, and in particular,orthologs, paralogs or nonorthologous gene displacements.

An ortholog is a gene or genes that are related by vertical descent andare responsible for substantially the same or identical functions indifferent organisms. For example, mouse epoxide hydrolase and humanepoxide hydrolase can be considered orthologs for the biologicalfunction of hydrolysis of epoxides. Genes are related by verticaldescent when, for example, they share sequence similarity of sufficientamount to indicate they are homologous, or related by evolution from acommon ancestor. Genes can also be considered orthologs if they sharethree-dimensional structure but not necessarily sequence similarity, ofa sufficient amount to indicate that they have evolved from a commonancestor to the extent that the primary sequence similarity is notidentifiable. Genes that are orthologous can encode proteins withsequence similarity of about 25% to 100% amino acid sequence identity.Genes encoding proteins sharing an amino acid similarity less that 25%can also be considered to have arisen by vertical descent if theirthree-dimensional structure also shows similarities. Members of theserine protease family of enzymes, including tissue plasminogenactivator and elastase, are considered to have arisen by verticaldescent from a common ancestor.

Orthologs include genes or their encoded gene products that through, forexample, evolution, have diverged in structure or overall activity. Forexample, where one species encodes a gene product exhibiting twofunctions and where such functions have been separated into distinctgenes in a second species, the three genes and their correspondingproducts are considered to be orthologs. For the production of abiochemical product, those skilled in the art will understand that theorthologous gene harboring the metabolic activity to be introduced ordisrupted is to be chosen for construction of the non-naturallyoccurring microorganism. An example of orthologs exhibiting separableactivities is where distinct activities have been separated intodistinct gene products between two or more species or within a singlespecies. A specific example is the separation of elastase proteolysisand plasminogen proteolysis, two types of serine protease activity, intodistinct molecules as plasminogen activator and elastase. A secondexample is the separation of mycoplasma 5′-3′ exonuclease and DrosophilaDNA polymerase III activity. The DNA polymerase from the first speciescan be considered an ortholog to either or both of the exonuclease orthe polymerase from the second species and vice versa.

In contrast, paralogs are homologs related by, for example, duplicationfollowed by evolutionary divergence and have similar or common, but notidentical functions. Paralogs can originate or derive from, for example,the same species or from a different species. For example, microsomalepoxide hydrolase (epoxide hydrolase I) and soluble epoxide hydrolase(epoxide hydrolase II) can be considered paralogs because they representtwo distinct enzymes, co-evolved from a common ancestor, that catalyzedistinct reactions and have distinct functions in the same species.Paralogs are proteins from the same species with significant sequencesimilarity to each other suggesting that they are homologous, or relatedthrough co-evolution from a common ancestor. Groups of paralogousprotein families include HipA homologs, luciferase genes, peptidases,and others.

A nonorthologous gene displacement is a nonorthologous gene from onespecies that can substitute for a referenced gene function in adifferent species. Substitution includes, for example, being able toperform substantially the same or a similar function in the species oforigin compared to the referenced function in the different species.Although generally, a nonorthologous gene displacement will beidentifiable as structurally related to a known gene encoding thereferenced function, less structurally related but functionally similargenes and their corresponding gene products nevertheless will still fallwithin the meaning of the term as it is used herein. Functionalsimilarity requires, for example, at least some structural similarity inthe active site or binding region of a nonorthologous gene productcompared to a gene encoding the function sought to be substituted.Therefore, a nonorthologous gene includes, for example, a paralog or anunrelated gene.

Therefore, in identifying and constructing the non-naturally occurringmicrobial organisms of the invention having 2H3M4OP, p-toluate orterephthalate biosynthetic capability, those skilled in the art willunderstand with applying the teaching and guidance provided herein to aparticular species that the identification of metabolic modificationscan include identification and inclusion or inactivation of orthologs.To the extent that paralogs and/or nonorthologous gene displacements arepresent in the referenced microorganism that encode an enzyme catalyzinga similar or substantially similar metabolic reaction, those skilled inthe art also can utilize these evolutionally related genes.

Orthologs, paralogs and nonorthologous gene displacements can bedetermined by methods well known to those skilled in the art. Forexample, inspection of nucleic acid or amino acid sequences for twopolypeptides will reveal sequence identity and similarities between thecompared sequences. Based on such similarities, one skilled in the artcan determine if the similarity is sufficiently high to indicate theproteins are related through evolution from a common ancestor.Algorithms well known to those skilled in the art, such as Align, BLAST,Clustal W and others compare and determine a raw sequence similarity oridentity, and also determine the presence or significance of gaps in thesequence which can be assigned a weight or score. Such algorithms alsoare known in the art and are similarly applicable for determiningnucleotide sequence similarity or identity. Parameters for sufficientsimilarity to determine relatedness are computed based on well knownmethods for calculating statistical similarity, or the chance of findinga similar match in a random polypeptide, and the significance of thematch determined. A computer comparison of two or more sequences can, ifdesired, also be optimized visually by those skilled in the art. Relatedgene products or proteins can be expected to have a high similarity, forexample, 25% to 100% sequence identity. Proteins that are unrelated canhave an identity which is essentially the same as would be expected tooccur by chance, if a database of sufficient size is scanned (about 5%).Sequences between 5% and 24% may or may not represent sufficienthomology to conclude that the compared sequences are related. Additionalstatistical analysis to determine the significance of such matches giventhe size of the data set can be carried out to determine the relevanceof these sequences.

Exemplary parameters for determining relatedness of two or moresequences using the BLAST algorithm, for example, can be as set forthbelow. Briefly, amino acid sequence alignments can be performed usingBLASTP version 2.0.8 (Jan. 5, 1999) and the following parameters:Matrix: 0 BLOSUM62; gap open: 11; gap extension: 1; x_dropoff: 50;expect: 10.0; wordsize: 3; filter: on. Nucleic acid sequence alignmentscan be performed using BLASTN version 2.0.6 (Sep. 16, 1998) and thefollowing parameters: Match: 1; mismatch: −2; gap open: 5; gapextension: 2; x_dropoff: 50; expect: 10.0; wordsize: 11; filter: off.Those skilled in the art will know what modifications can be made to theabove parameters to either increase or decrease the stringency of thecomparison, for example, and determine the relatedness of two or moresequences.

In one embodiment, the invention provides a non-naturally occurringmicrobial organism having a 2H3M4OP pathway and including at least oneexogenous nucleic acid encoding a 2H3M4OP pathway enzyme expressed in asufficient amount to produce 2H3M4OP. The 2H3M4OP pathway of themicrobial organism can include a pathway selected from: (1) 1A, 1B, 1C,1D, 1E and 1F; (2) 2A, 2B and 2C; and (3) 2D, 2E and 2C, wherein 1A isan erythrose-4-phosphate dehydrogenase, wherein 1B is a4-phosphoerythronate dehydrogenase, wherein 1C is a2-acetyl-2,3-dihydroxy-4-phosphobutanoate synthase, wherein 1D is a2-acetyl-2,3-dihydroxy-4-phosphobutanoate reductoisomerase, wherein 1Eis a 2,3,4-trihydroxy-3-methyl-5-phosphopentanoate dehydratase, wherein1F is a 4-hydroxy-3-methyl-2-oxo-5-phosphopentanoate decarboxylase,wherein 2A is a 4,5-dihydroxy-2-oxopentanoate methyltransferase, wherein2B is a 4,5-dihydroxy-3-methyl-2-oxopentanoate kinase, wherein 2C is a4-hydroxy-3-methyl-2-oxo-5-phosphopentanoate decarboxylase, wherein 2Dis a 4,5-dihydroxy-2-oxopentanoate kinase, wherein 2E is a4-hydroxy-2-oxo-5-phosphopentanoate methyltransferase (see FIGS. 1 and 2and Example I). Additionally, in some aspects, the microbial organism ofthe invention can include two, three, four, five or six exogenousnucleic acids, wherein each exogenous nucleic acid encodes a 2H3M4OPpathway enzyme as described herein. In some aspects, the inventionprovides a microbial organism of the invention having exogenous nucleicacids encoding each of the 2H3M4OP pathway enzymes of at least one ofthe 2H3M4OP pathways selected from (1)-(3), as described above.

In some aspects, the invention provides the 2H3M4OP pathway of themicrobial organisms includes a 2-C-methyl-D-erythritol-4-phosphatedehydratase (see Example II and FIG. 3, step C). A non-naturallyoccurring microbial organism having a 2H3M4OP pathway can further have a1-deoxyxylulose-5-phosphate synthase or a 1-deoxy-D-xylulose-5-phosphatereductoisomerase (see Example II and FIG. 3, steps A and B). Thus, a2H3M4OP pathway can include a 2-C-methyl-D-erythritol-4-phosphatedehydratase, a 1-deoxyxylulose-5-phosphate synthase and a1-deoxy-D-xylulose-5-phosphate reductoisomerase.

The invention also provides a non-naturally occurring microbial organismhaving a 2H3M4OP pathway as described herein and/or a p-toluate pathway.In this aspect, the p-toluate pathway can include at least one exogenousnucleic acid encoding a p-toluate pathway enzyme expressed in asufficient amount to produce p-toluate. In some aspects, the p-toluatepathway includes 4A, 4B, 4C, 4D, 4E, 4F, 4G and/or 4H, wherein 4A is a2-dehydro-3-deoxyphosphoheptonate synthase; wherein 4B is a3-dehydroquinate synthase; wherein 4C is a 3-dehydroquinate dehydratase;wherein 4D is a shikimate dehydrogenase; wherein 4E is a shikimatekinase; wherein 4F is a 3-phosphoshikimate-2-carboxyvinyltransferase;wherein 4G is a chorismate synthase and wherein 4H is a chorismate lyase(see Example III and FIG. 4, steps A-H). A non-naturally occurringmicrobial organism having a p-toluate pathway can further include a2H3M4OP pathway as described herein (see Examples I and II and FIGS.1-3). For example, a 2H3M4OP pathway can include a pathway selectedfrom: (1) 1A, 1B, 1C, 1D, 1E and 1F; (2) 2A, 2B and 2C; and (3) 2D, 2Eand 2C, wherein 1A is an erythrose-4-phosphate dehydrogenase, wherein 1Bis a 4-phosphoerythronate dehydrogenase, wherein 1C is a2-acetyl-2,3-dihydroxy-4-phosphobutanoate synthase, wherein 1D is a2-acetyl-2,3-dihydroxy-4-phosphobutanoate reductoisomerase, wherein 1Eis a 2,3,4-trihydroxy-3-methyl-5-phosphopentanoate dehydratase, wherein1F is a 4-hydroxy-3-methyl-2-oxo-5-phosphopentanoate decarboxylase,wherein 2A is a 4,5-dihydroxy-2-oxopentanoate methyltransferase, wherein2B is a 4,5-dihydroxy-3-methyl-2-oxopentanoate kinase, wherein 2C is a4-hydroxy-3-methyl-2-oxo-5-phosphopentanoate decarboxylase, wherein 2Dis a 4,5-dihydroxy-2-oxopentanoate kinase, wherein 2E is a4-hydroxy-2-oxo-5-phosphopentanoate methyltransferase (see FIGS. 1 and 2and Example I). Alternatively, a 2H3M4OP pathway can include a2-C-methyl-D-erythritol-4-phosphate dehydratase, a1-deoxyxylulose-5-phosphate synthase and/or a1-deoxy-D-xylulose-5-phosphate reductoisomerase (see FIG. 3 and ExampleII).

In some aspects, the microbial organism of the invention includes two,three, four, five, six, seven or eight exogenous nucleic acids, whereineach nucleic acid encodes a p-toluate pathway enzyme. Additionally, insome aspects, the invention provides that the microbial organism of theinvention includes exogenous nucleic acids encoding each of the enzymesof the p-toluate pathway disclosed herein.

The invention additionally provides a non-naturally occurring microbialorganism having a 2H3M4OP pathway and/or a p-toluate pathway asdisclosed herein and/or a terephthalate pathway. In this aspect, theterephthalate pathway can include at least one exogenous nucleic acidencoding a terephthalate pathway enzyme expressed in a sufficient amountto produce terephthalate. In some aspects of the invention, theterephthalate pathway can include 5A, 5B and 5C, wherein 5A is ap-toluate methyl-monooxygenase reductase, wherein 5B is a4-carboxybenzyl alcohol dehydrogenase and wherein 5C is a4-carboxybenzyl aldehyde dehydrogenase (see Example IV and FIG. 5). Suchan organism containing a terephthalate pathway can additionally includea p-toluate pathway, wherein the p-toluate pathway includes 4A, 4B, 4C,4D, 4E, 4F, 4G and/or 4H, wherein 4A is a2-dehydro-3-deoxyphosphoheptonate synthase; wherein 4B is a3-dehydroquinate synthase; wherein 4C is a 3-dehydroquinate dehydratase;wherein 4D is a shikimate dehydrogenase; wherein 4E is a shikimatekinase; wherein 4F is a 3-phosphoshikimate-2-carboxyvinyltransferase;wherein 4G is a chorismate synthase and wherein 4H is a chorismate lyase(see Examples III and IV and FIGS. 4 and 5). Such a non-naturallyoccurring microbial organism having a terephthalate pathway and ap-toluate pathway can further include a 2H3M4OP pathway as describedherein (see Examples I and II and FIGS. 1-3). For example, a 2H3M4OPpathway can include a pathway selected from: (1) 1A, 1B, 1C, 1D, 1E and1F; (2) 2A, 2B and 2C; and (3) 2D, 2E and 2C, wherein 1A is anerythrose-4-phosphate dehydrogenase, wherein 1B is a4-phosphoerythronate dehydrogenase, wherein 1C is a2-acetyl-2,3-dihydroxy-4-phosphobutanoate synthase, wherein 1D is a2-acetyl-2,3-dihydroxy-4-phosphobutanoate reductoisomerase, wherein 1Eis a 2,3,4-trihydroxy-3-methyl-5-phosphopentanoate dehydratase, wherein1F is a 4-hydroxy-3-methyl-2-oxo-5-phosphopentanoate decarboxylase,wherein 2A is a 4,5-dihydroxy-2-oxopentanoate methyltransferase, wherein2B is a 4,5-dihydroxy-3-methyl-2-oxopentanoate kinase, wherein 2C is a4-hydroxy-3-methyl-2-oxo-5-phosphopentanoate decarboxylase, wherein 2Dis a 4,5-dihydroxy-2-oxopentanoate kinase, wherein 2E is a4-hydroxy-2-oxo-5-phosphopentanoate methyltransferase (see FIGS. 1 and 2and Example I). Alternatively, a 2H3M4OP pathway can include a2-C-methyl-D-erythritol-4-phosphate dehydratase, a1-deoxyxylulose-5-phosphate synthase and/or a1-deoxy-D-xylulose-5-phosphate reductoisomerase (see FIG. 3 and ExampleII).

In some aspects, the microbial organism of the invention includes two orthree exogenous nucleic acids, wherein each nucleic acid encodes aterephthalate pathway enzyme. Additionally, in some aspects, theinvention provides that the microbial organism of the invention includesexogenous nucleic acids encoding each of the enzymes of theterephthalate pathway disclosed herein.

In an additional embodiment, the invention provides a non-naturallyoccurring microbial organism having a 2H3M4OP, p-toluate and/orterephthalate pathway, wherein the non-naturally occurring microbialorganism comprises at least one exogenous nucleic acid encoding anenzyme or protein that converts a substrate to a product. For example,in a 2H3M4OP pathway, the substrates and products can be selected fromthe group consisting of erythrose-4-phosphate to 4-phosphoerythronate;4-phosphoerythronate to 2-oxo-3-hydroxy-4-phosphobutanoate;2-oxo-3-hydroxy-4-phosphobutanoate to2-acetyl-2,3-dihydroxy-4-phosphobutanoate;2-acetyl-2,3-dihydroxy-4-phosphobutanoate to2,3,4-trihydroxy-3-methyl-5-phosphopentanoate;2,3,4-trihydroxy-3-methyl-5-phosphopentanoate to4-hydroxy-3-methyl-2-oxo-5-phosphopentanoate;4-hydroxy-3-methyl-2-oxo-5-phosphopentanoate to(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate;4,5-dihydroxy-2-oxopentanoate to 4,5-dihydroxy-3-methyl-2-oxopentanoate;4,5-dihydroxy-2-oxopentanoate to 4-hydroxy-2-oxo-5-phosphopentanoate;4-hydroxy-2-oxo-5-phosphopentanoate to4-hydroxy-3-methyl-2-oxo-5-phosphopentanoate;4,5-dihydroxy-3-methyl-2-oxopentanoate to4-hydroxy-3-methyl-2-oxo-5-phosphopentanoate; glyceraldehyde-3-phosphateand pyruvate to 1-deoxy-D-xylulose-5-phosphate;1-deoxy-D-xylulose-5-phosphate to C-methyl-D-erythritol-4-phosphate; andC-methyl-D-erythritol-4-phosphate to(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate (see Examples I and II andFIGS. 1-3). In another embodiment, a p-toluate pathway can comprisesubstrates and products selected from(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate to2,4-dihydroxy-5-methyl-6-[(phosphonooxy)methyl]oxane-2-carboxylate;2,4-dihydroxy-5-methyl-6-[(phosphonooxy)methyl]oxane-2-carboxylate to1,3-dihydroxy-4-methyl-5-oxocyclohexane-1-carboxylate;1,3-dihydroxy-4-methyl-5-oxocyclohexane-1-carboxylate to5-hydroxy-4-methyl-3-oxocyclohex-1-ene-1-carboxylic acid;5-hydroxy-4-methyl-3-oxocyclohex-1-ene-1-carboxylic acid to3,5-dihydroxy-4-methylcyclohex-1-ene-1-carboxylate;3,5-dihydroxy-4-methylcyclohex-1-ene-1-carboxylate to5-hydroxy-4-methyl-3-(phosphonooxy)cyclohex-1-ene-1-carboxylate;5-hydroxy-4-methyl-3-(phosphonooxy)cyclohex-1-ene-1-carboxylate to5-[(1-carboxyeth-1-en-1-yl)oxy]-4-methyl-3-(phosphonooxy)cyclohex-1-ene-1-carboxylate;5-[(1-carboxyeth-1-en-1-yl)oxy]-4-methyl-3-(phosphonooxy)cyclohex-1-ene-1-carboxylateto3-[(1-carboxyeth-1-en-1-yl)oxy]-4-methylcyclohexa-1,5-diene-1-carboxylate;and3-[(1-carboxyeth-1-en-1-yl)oxy]-4-methylcyclohexa-1,5-diene-1-carboxylateto p-toluate (see Example II and FIG. 2). In still another embodiment, aterephthalate pathway can comprise substrates and products selected fromp-toluate to 4-carboxybenzyl alcohol; 4-carboxybenzyl alcohol to4-carboxybenzaldehyde; and 4-carboxybenzaldehyde to and terephthalicacid (see Example III and FIG. 3). One skilled in the art willunderstand that these are merely exemplary and that any of thesubstrate-product pairs disclosed herein suitable to produce a desiredproduct and for which an appropriate activity is available for theconversion of the substrate to the product can be readily determined byone skilled in the art based on the teachings herein. Thus, theinvention provides a non-naturally occurring microbial organismcontaining at least one exogenous nucleic acid encoding an enzyme orprotein, where the enzyme or protein converts the substrates andproducts of a 2H3M4OP, p-toluate or terephthalate pathway, such as thatshown in FIGS. 1-5.

While generally described herein as a microbial organism that contains a2H3M4OP, p-toluate or terephthalate pathway, it is understood that theinvention additionally provides a non-naturally occurring microbialorganism comprising at least one exogenous nucleic acid encoding a2H3M4OP, p-toluate or terephthalate pathway enzyme expressed in asufficient amount to produce an intermediate of a 2H3M4OP, p-toluate orterephthalate pathway. For example, as disclosed herein, a 2H3M4OP,p-toluate or terephthalate pathway is exemplified in FIGS. 1-5.Therefore, in addition to a microbial organism containing a 2H3M4OP,p-toluate or terephthalate pathway that produces 2H3M4OP, p-toluate orterephthalate, the invention additionally provides a non-naturallyoccurring microbial organism comprising at least one exogenous nucleicacid encoding a 2H3M4OP, p-toluate or terephthalate pathway enzyme,where the microbial organism produces a 2H3M4OP, p-toluate orterephthalate pathway intermediate, for example, 4-phosphoerythronate,2-oxo-3-hydroxy-4-phosphobutanoate, 2-acetyl-2,3-phosphodutanoate,2-acetyl-2,3-dihydroxy-4-phosphobutanoate,2,3,4-trihydroxy-3-methyl-5-phosphopentanoate,4-hydroxy-3-methyl-2-oxo-5-phosphopentanoate,4,5-dihydroxy-3-methyl-2-oxopentanoate,4-hydroxy-2-oxo-5-phosphopentanoate, 1-deoxy-D-xylulose-5-phosphate,C-methyl-D-erythritol-4-phosphate,2,4-dihydroxy-5-methyl-6-[(phosphonooxy)methyl]oxane-2-carboxylate,1,3-dihydroxy-4-methyl-5-oxocyclohexane-1-carboxylate,5-hydroxy-4-methyl-3-oxocyclohex-1-ene-1-carboxylate,3,5-dihydroxy-4-methylcyclohex-1-ene-1-carboxylate,5-hydroxy-4-methyl-3-(phosphonooxy)cyclohex-1-ene-1-carboxylate,5-[(1-carboxyeth-1-en-1-yl)oxy]-4-methyl-3-(phosphonooxy)cyclohex-1-ene-1-carboxylate,3-[(1-carboxyeth-1-en-1-yl)oxy]-4-methylcyclohexa-1,5-diene-1-carboxylate,4-carboxybenzyl alcohol or 4-carboxybenzaldehyde.

It is understood that any of the pathways disclosed herein, as describedin the Examples and exemplified in the Figures, including the pathwaysof FIGS. 1-5, can be utilized to generate a non-naturally occurringmicrobial organism that produces any pathway intermediate or product, asdesired. As disclosed herein, such a microbial organism that produces anintermediate can be used in combination with another microbial organismexpressing downstream pathway enzymes to produce a desired product.However, it is understood that a non-naturally occurring microbialorganism that produces a 2H3M4OP, p-toluate or terephthalate pathwayintermediate can be utilized to produce the intermediate as a desiredproduct.

The invention is described herein with general reference to themetabolic reaction, reactant or product thereof, or with specificreference to one or more nucleic acids or genes encoding an enzymeassociated with or catalyzing, or a protein associated with, thereferenced metabolic reaction, reactant or product. Unless otherwiseexpressly stated herein, those skilled in the art will understand thatreference to a reaction also constitutes reference to the reactants andproducts of the reaction. Similarly, unless otherwise expressly statedherein, reference to a reactant or product also references the reaction,and reference to any of these metabolic constituents also references thegene or genes encoding the enzymes that catalyze or proteins involved inthe referenced reaction, reactant or product. Likewise, given the wellknown fields of metabolic biochemistry, enzymology and genomics,reference herein to a gene or encoding nucleic acid also constitutes areference to the corresponding encoded enzyme and the reaction itcatalyzes or a protein associated with the reaction as well as thereactants and products of the reaction.

As disclosed herein, the product p-toluate or terephthalate or theintermediate 4-phosphoerythronate, 2-oxo-3-hydroxy-4-phosphobutanoate,2-acetyl-2,3-phosphodutanoate,2-acetyl-2,3-dihydroxy-4-phosphobutanoate,2,3,4-trihydroxy-3-methyl-5-phosphopentanoate,4-hydroxy-3-methyl-2-oxo-5-phosphopentanoate,4,5-dihydroxy-3-methyl-2-oxopentanoate,4-hydroxy-2-oxo-5-phosphopentanoate,2,4-dihydroxy-5-methyl-6-[(phosphonooxy)methyl]oxane-2-carboxylate,1,3-dihydroxy-4-methyl-5-oxocyclohexane-1-carboxylate,5-hydroxy-4-methyl-3-oxocyclohex-1-ene-1-carboxylate,3,5-dihydroxy-4-methylcyclohex-1-ene-1-carboxylate,5-hydroxy-4-methyl-3-(phosphonooxy)cyclohex-1-ene-1-carboxylate,5-[(1-carboxyeth-1-en-1-yl)oxy]-4-methyl-3-(phosphonooxy)cyclohex-1-ene-1-carboxylate,3-[(1-carboxyeth-1-en-1-yl)oxy]-4-methylcyclohexa-1,5-diene-1-carboxylate,4-carboxybenzyl alcohol or 4-carboxybenzaldehyde, as well as otherintermediates, are carboxylic acids, which can occur in various ionizedforms, including fully protonated, partially protonated, and fullydeprotonated forms. Accordingly, the suffix “-ate,” or the acid form,can be used interchangeably to describe both the free acid form as wellas any deprotonated form, in particular since the ionized form is knownto depend on the pH in which the compound is found. It is understoodthat carboxylate products or intermediates includes ester forms ofcarboxylate products or pathway intermediates, such as O-carboxylate andS-carboxylate esters. O- and S-carboxylates can include lower alkyl,that is C1 to C6, branched or straight chain carboxylates. Some such O-or S-carboxylates include, without limitation, methyl, ethyl, n-propyl,n-butyl, i-propyl, sec-butyl, and tert-butyl, pentyl, hexyl O- orS-carboxylates, any of which can further possess an unsaturation,providing for example, propenyl, butenyl, pentyl, and hexenyl O- orS-carboxylates. O-carboxylates can be the product of a biosyntheticpathway. Exemplary O-carboxylates accessed via biosynthetic pathways caninclude, without limitation, methyl terephthalate, ethyl terephthalate,and n-propyl terephthalate. Other biosynthetically accessibleO-carboxylates can include medium to long chain groups, that is C7-C22,O-carboxylate esters derived from fatty alcohols, such heptyl, octyl,nonyl, decyl, undecyl, lauryl, tridecyl, myristyl, pentadecyl, cetyl,palmitolyl, heptadecyl, stearyl, nonadecyl, arachidyl, heneicosyl, andbehenyl alcohols, any one of which can be optionally branched and/orcontain unsaturations. O-carboxylate esters can also be accessed via abiochemical or chemical process, such as esterification of a freecarboxylic acid product or transesterification of an O- orS-carboxylate. S-carboxylates are exemplified by CoA S-esters, cysteinylS-esters, alkylthioesters, and various aryl and heteroaryl thioesters.

The non-naturally occurring microbial organisms of the invention can beproduced by introducing expressible nucleic acids encoding one or moreof the enzymes or proteins participating in one or more 2H3M4OP,p-toluate or terephthalate biosynthetic pathways. Depending on the hostmicrobial organism chosen for biosynthesis, nucleic acids for some orall of a particular 2H3M4OP, p-toluate or terephthalate biosyntheticpathway can be expressed. For example, if a chosen host is deficient inone or more enzymes or proteins for a desired biosynthetic pathway, thenexpressible nucleic acids for the deficient enzyme(s) or protein(s) areintroduced into the host for subsequent exogenous expression.Alternatively, if the chosen host exhibits endogenous expression of somepathway genes, but is deficient in others, then an encoding nucleic acidis needed for the deficient enzyme(s) or protein(s) to achieve 2H3M4OP,p-toluate or terephthalate biosynthesis. Thus, a non-naturally occurringmicrobial organism of the invention can be produced by introducingexogenous enzyme or protein activities to obtain a desired biosyntheticpathway or a desired biosynthetic pathway can be obtained by introducingone or more exogenous enzyme or protein activities that, together withone or more endogenous enzymes or proteins, produces a desired productsuch as 2H3M4OP, p-toluate or terephthalate.

Host microbial organisms can be selected from, and the non-naturallyoccurring microbial organisms generated in, for example, bacteria,yeast, fungus or any of a variety of other microorganisms applicable orsuitable to fermentation processes. Exemplary bacteria include anyspecies selected from the order Enterobacteriales, familyEnterobacteriaceae, including the genera Escherichia and Klebsiella; theorder Aeromonadales, family Succinivibrionaceae, including the genusAnaerobiospirillum; the order Pasteurellales, family Pasteurellaceae,including the genera Actinobacillus and Mannheimia; the orderRhizobiales, family Bradyrhizobiaceae, including the genus Rhizobium;the order Bacillales, family Bacillaceae, including the genus Bacillus;the order Actinomycetales, families Corynebacteriaceae andStreptomycetaceae, including the genus Corynebacterium and the genusStreptomyces, respectively; order Rhodospirillales, familyAcetobacteraceae, including the genus Gluconobacter; the orderSphingomonadales, family Sphingomonadaceae, including the genusZymomonas; the order Lactobacillales, families Lactobacillaceae andStreptococcaceae, including the genus Lactobacillus and the genusLactococcus, respectively; the order Clostridiales, familyClostridiaceae, genus Clostridium; and the order Pseudomonadales, familyPseudomonadaceae, including the genus Pseudomonas. Non-limiting speciesof host bacteria include Escherichia coli, Klebsiella oxytoca,Anaerobiospirillum succiniciproducens, Actinobacillus succinogenes,Mannheimia succiniciproducens, Rhizobium etli, Bacillus subtilis,Corynebacterium glutamicum, Gluconobacter oxydans, Zymomonas mobilis,Lactococcus lactis, Lactobacillus plantarum, Streptomyces coelicolor,Clostridium acetobutylicum, Pseudomonas fluorescens, and Pseudomonasputida.

Similarly, exemplary species of yeast or fungi species include anyspecies selected from the order Saccharomycetales, familySaccaromycetaceae, including the genera Saccharomyces, Kluyveromyces andPichia; the order Saccharomycetales, family Dipodascaceae, including thegenus Yarrowia; the order Schizosaccharomycetales, familySchizosaccaromycetaceae, including the genus Schizosaccharomyces; theorder Eurotiales, family Trichocomaceae, including the genusAspergillus; and the order Mucorales, family Mucoraceae, including thegenus Rhizopus. Non-limiting species of host yeast or fungi includeSaccharomyces cerevisiae, Schizosaccharomyces pombe, Candida albicans,Kluyveromyces lactis, Kluyveromyces marxianus, Aspergillus terreus,Aspergillus niger, Pichia pastoris, Rhizopus arrhizus, Rhizopus oryzae,Yarrowia lipolytica, and the like. Preferred host microbial organismscan be selected from, and the non-naturally occurring microbialorganisms generated in, for example, bacteria, yeast, fungus or any of avariety of other microorganisms applicable to fermentation processes, asdisclosed in U.S. Patent Publication No. US 2011/0207185 A1. E. coli isa particularly useful and preferred host organism since it is a wellcharacterized microbial organism suitable for genetic engineering. Otherparticularly useful host organisms include yeast such as Saccharomycescerevisiae. It is understood that any suitable microbial host organismcan be used to introduce metabolic and/or genetic modifications toproduce a desired product.

Depending on the 2H3M4OP, p-toluate or terephthalate biosyntheticpathway constituents of a selected host microbial organism, thenon-naturally occurring microbial organisms of the invention willinclude at least one exogenously expressed 2H3M4OP, p-toluate orterephthalate pathway-encoding nucleic acid and up to all encodingnucleic acids for one or more 2H3M4OP, p-toluate or terephthalatebiosynthetic pathways. For example, 2H3M4OP, p-toluate or terephthalatebiosynthesis can be established in a host deficient in a pathway enzymeor protein through exogenous expression of the corresponding encodingnucleic acid. In a host deficient in all enzymes or proteins of a2H3M4OP, p-toluate or terephthalate pathway, exogenous expression of allenzyme or proteins in the pathway can be included, although it isunderstood that all enzymes or proteins of a pathway can be expressedeven if the host contains at least one of the pathway enzymes orproteins. For example, exogenous expression of all enzymes or proteinsin a pathway for production of 2H3M4OP, p-toluate or terephthalate canbe included. A non-limiting example of all enzymes in a p-toluatepathway includes a 2-dehydro-3-deoxyphosphoheptonate synthase; a3-dehydroquinate synthase; a 3-dehydroquinate dehydratase; a shikimatedehydrogenase; shikimate kinase; a3-phosphoshikimate-2-carboxyvinyltransferase; a chorismate synthase; anda chorismate lyase. In addition, a non-limiting example of all enzymesin a terephthalate pathway included a p-toluate methyl-monooxygenasereductase; a 4-carboxybenzyl alcohol dehydrogenase; and a4-carboxybenzyl aldehyde dehydrogenase. Furthermore, a non-limitingexample of all enzymes in a 2H3M4OP pathway include anerythrose-4-phosphate dehydrogenase, a 4-phosphoerythronatedehydrogenase, a 2-acetyl-2,3-dihydroxy-4-phosphobutanoate synthase, a2-acetyl-2,3-dihydroxy-4-phosphobutanoate reductoisomerase, a2,3,4-trihydroxy-3-methyl-5-phosphopentanoate dehydratase and a4-hydroxy-3-methyl-2-oxo-5-phosphopentanoate decarboxylase.

Given the teachings and guidance provided herein, those skilled in theart will understand that the number of encoding nucleic acids tointroduce in an expressible form will, at least, parallel the 2H3M4OP,p-toluate or terephthalate pathway deficiencies of the selected hostmicrobial organism. Therefore, a non-naturally occurring microbialorganism of the invention can have one, two, three, four, five, six,seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen,sixteen or seventeen, up to all nucleic acids encoding the enzymes orproteins constituting a 2H3M4OP, p-toluate or terephthalate biosyntheticpathway disclosed herein. In some embodiments, the non-naturallyoccurring microbial organisms also can include other geneticmodifications that facilitate or optimize 2H3M4OP, p-toluate orterephthalate biosynthesis or that confer other useful functions ontothe host microbial organism. One such other functionality can include,for example, augmentation of the synthesis of one or more of the2H3M4OP, p-toluate or terephthalate pathway precursors such aserythrose-4-phosphate, 4,5-dihydroxy-2-oxopentanoate,glyceraldehyde-3-phosphate and pyruvate.

Generally, a host microbial organism is selected such that it producesthe precursor of a 2H3M4OP, p-toluate or terephthalate pathway, eitheras a naturally produced molecule or as an engineered product that eitherprovides de novo production of a desired precursor or increasedproduction of a precursor naturally produced by the host microbialorganism. For example, erythrose-4-phosphate,4,5-dihydroxy-2-oxopentanoate, glyceraldehyde-3-phosphate and pyruvateare produced naturally in a host organism such as E. coli. A hostorganism can be engineered to increase production of a precursor, asdisclosed herein. In addition, a microbial organism that has beenengineered to produce a desired precursor can be used as a host organismand further engineered to express enzymes or proteins of a 2H3M4OP,p-toluate or terephthalate pathway.

In some embodiments, a non-naturally occurring microbial organism of theinvention is generated from a host that contains the enzymaticcapability to synthesize 2H3M4OP, p-toluate or terephthalate. In thisspecific embodiment it can be useful to increase the synthesis oraccumulation of a 2H3M4OP, p-toluate or terephthalate pathway productto, for example, drive 2H3M4OP, p-toluate or terephthalate pathwayreactions toward 2H3M4OP, p-toluate or terephthalate production.Increased synthesis or accumulation can be accomplished by, for example,overexpression of nucleic acids encoding one or more of theabove-described 2H3M4OP, p-toluate or terephthalate pathway enzymes orproteins. Overexpression of the enzyme or enzymes and/or protein orproteins of the 2H3M4OP, p-toluate or terephthalate pathway can occur,for example, through exogenous expression of the endogenous gene orgenes, or through exogenous expression of the heterologous gene orgenes. Therefore, naturally occurring organisms can be readily generatedto be non-naturally occurring microbial organisms of the invention, forexample, producing 2H3M4OP, p-toluate or terephthalate, throughoverexpression of one, two, three, four, five one, two, three, four,five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen,fifteen, sixteen or seventeen, that is, up to all nucleic acids encoding2H3M4OP, p-toluate or terephthalate biosynthetic pathway enzymes orproteins. In addition, a non-naturally occurring organism can begenerated by mutagenesis of an endogenous gene that results in anincrease in activity of an enzyme in the 2H3M4OP, p-toluate orterephthalate biosynthetic pathway.

In particularly useful embodiments, exogenous expression of the encodingnucleic acids is employed. Exogenous expression confers the ability tocustom tailor the expression and/or regulatory elements to the host andapplication to achieve a desired expression level that is controlled bythe user. However, endogenous expression also can be utilized in otherembodiments such as by removing a negative regulatory effector orinduction of the gene's promoter when linked to an inducible promoter orother regulatory element. Thus, an endogenous gene having a naturallyoccurring inducible promoter can be up-regulated by providing theappropriate inducing agent, or the regulatory region of an endogenousgene can be engineered to incorporate an inducible regulatory element,thereby allowing the regulation of increased expression of an endogenousgene at a desired time. Similarly, an inducible promoter can be includedas a regulatory element for an exogenous gene introduced into anon-naturally occurring microbial organism.

It is understood that, in methods of the invention, any of the one ormore exogenous nucleic acids can be introduced into a microbial organismto produce a non-naturally occurring microbial organism of theinvention. The nucleic acids can be introduced so as to confer, forexample, a 2H3M4OP, p-toluate or terephthalate biosynthetic pathway ontothe microbial organism. Alternatively, encoding nucleic acids can beintroduced to produce an intermediate microbial organism having thebiosynthetic capability to catalyze some of the required reactions toconfer 2H3M4OP, p-toluate or terephthalate biosynthetic capability. Forexample, a non-naturally occurring microbial organism having a 2H3M4OP,p-toluate or terephthalate biosynthetic pathway can comprise at leasttwo exogenous nucleic acids encoding desired enzymes or proteins, suchas the combination of a erythrose-4-phosphate dehydrogenase and a4-phosphoerythronate dehydrogenase, or alternatively a2-acetyl-2,3-dihydroxy-4-phosphobutanoate reductoisomerase and a4-hydroxy-3-methyl-2-oxo-5-phosphopentanoate decarboxylase, oralternatively a 4,5-dihydroxy-3-methyl-2-oxopentanoate kinase and a4-hydroxy-3-methyl-2-oxo-5-phosphopentanoate decarboxylase, oralternatively a 2-dehydro-3-deoxyphosphoheptonate synthase and ashikimate kinase, or alternatively a 4-carboxybenzyl alcoholdehydrogenase and a 4-carboxybenzyl aldehyde dehydrogenase, and thelike. Thus, it is understood that any combination of two or more enzymesor proteins of a biosynthetic pathway can be included in a non-naturallyoccurring microbial organism of the invention. Similarly, it isunderstood that any combination of three or more enzymes or proteins ofa biosynthetic pathway can be included in a non-naturally occurringmicrobial organism of the invention, for example, a2-acetyl-2,3-dihydroxy-4-phosphobutanoate synthase, a2-acetyl-2,3-dihydroxy-4-phosphobutanoate reductoisomerase and a2,3,4-trihydroxy-3-methyl-5-phosphopentanoate dehydratase, oralternatively a 4-hydroxy-3-methyl-2-oxo-5-phosphopentanoatedecarboxylase, a 4,5-dihydroxy-2-oxopentanoate kinase and a4-hydroxy-2-oxo-5-phosphopentanoate methyltransferase, or alternativelya shikimate dehydrogenase, a shikimate kinase and a3-phosphoshikimate-2-carboxyvinyltransferase, and so forth, as desired,so long as the combination of enzymes and/or proteins of the desiredbiosynthetic pathway results in production of the corresponding desiredproduct. Similarly, any combination of four, five, six, seven, eight,nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen orseventeen or more enzymes or proteins of a biosynthetic pathway asdisclosed herein can be included in a non-naturally occurring microbialorganism of the invention, as desired, so long as the combination ofenzymes and/or proteins of the desired biosynthetic pathway results inproduction of the corresponding desired product.

In addition to the biosynthesis of 2H3M4OP, p-toluate or terephthalateas described herein, the non-naturally occurring microbial organisms andmethods of the invention also can be utilized in various combinationswith each other and with other microbial organisms and methods wellknown in the art to achieve product biosynthesis by other routes. Forexample, one alternative to produce 2H3M4OP, p-toluate or terephthalateother than use of the 2H3M4OP, p-toluate or terephthalate producers isthrough addition of another microbial organism capable of converting a2H3M4OP, p-toluate or terephthalate pathway intermediate to 2H3M4OP,p-toluate or terephthalate. One such procedure includes, for example,the fermentation of a microbial organism that produces a 2H3M4OP,p-toluate or terephthalate pathway intermediate. The 2H3M4OP, p-toluateor terephthalate pathway intermediate can then be used as a substratefor a second microbial organism that converts the 2H3M4OP, p-toluate orterephthalate pathway intermediate to 2H3M4OP, p-toluate orterephthalate. The 2H3M4OP, p-toluate or terephthalate pathwayintermediate can be added directly to another culture of the secondorganism or the original culture of the 2H3M4OP, p-toluate orterephthalate pathway intermediate producers can be depleted of thesemicrobial organisms by, for example, cell separation, and thensubsequent addition of the second organism to the fermentation broth canbe utilized to produce the final product without intermediatepurification steps.

In other embodiments, the non-naturally occurring microbial organismsand methods of the invention can be assembled in a wide variety ofsubpathways to achieve biosynthesis of, for example, 2H3M4OP, p-toluateor terephthalate. In these embodiments, biosynthetic pathways for adesired product of the invention can be segregated into differentmicrobial organisms, and the different microbial organisms can beco-cultured to produce the final product. In such a biosynthetic scheme,the product of one microbial organism is the substrate for a secondmicrobial organism until the final product is synthesized. For example,the biosynthesis of 2H3M4OP, p-toluate or terephthalate can beaccomplished by constructing a microbial organism that containsbiosynthetic pathways for conversion of one pathway intermediate toanother pathway intermediate or the product. Alternatively, 2H3M4OP,p-toluate or terephthalate also can be biosynthetically produced frommicrobial organisms through co-culture or co-fermentation using twoorganisms in the same vessel, where the first microbial organismproduces a 2H3M4OP intermediate, p-toluate intermediate or terephthalateintermediate and the second microbial organism converts the intermediateto 2H3M4OP, p-toluate or terephthalate.

Given the teachings and guidance provided herein, those skilled in theart will understand that a wide variety of combinations and permutationsexist for the non-naturally occurring microbial organisms and methods ofthe invention together with other microbial organisms, with theco-culture of other non-naturally occurring microbial organisms havingsubpathways and with combinations of other chemical and/or biochemicalprocedures well known in the art to produce 2H3M4OP, p-toluate orterephthalate.

Sources of encoding nucleic acids for a 2H3M4OP, p-toluate orterephthalate pathway enzyme or protein can include, for example, anyspecies where the encoded gene product is capable of catalyzing thereferenced reaction. Such species include both prokaryotic andeukaryotic organisms including, but not limited to, bacteria, includingarchaea and eubacteria, and eukaryotes, including yeast, plant, insect,animal, and mammal, including human. Exemplary species for such sourcesinclude, for example, Escherichia coli, Arabidopsis thaliana,Azospirillum brasilense, Bacillus subtilis, Bacteroides fragilis, Bostaurus, Bradyrhizobium japonicum USDA110, Burkholderia ambifaria,Burkholderia cenocepacia, Corynebacterium glutamicum, Homo sapiens,Lactococcus lactis, Mesorhizobium loti, Methanococcus aeolicus,Mycobacterium tuberculosis, Neurospora crassa, Pseudomonas aeruginosa,Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas stutzeri,Pseudomonas syringae, Ralstonia eutropha, Rattus norvegicus,Saccharomyces cerevisiae, Salmonella typhimurium, Sinorhizobiummelitoti, Streptomyces coelicolor, Streptomyces fradiae, Streptomycesluridus, Streptomyces roseosporus, Streptomyces viridochromogenes,Streptomyces wedmorensis, Sulfolobus solfataricus, Synechococcus sp. PCC7002, Thermoproteus tenax, Vibrio cholera, Xanthomonas oryzae andZymomonas mobilis, as well as other exemplary species disclosed hereinor available as source organisms for corresponding genes. Exemplaryspecies for such sources further include, for example, Escherichia coli,as well as other exemplary species disclosed in U.S. Patent PublicationNo. US 2011/0207185 A1. However, with the complete genome sequenceavailable for now more than 550 species (with more than half of theseavailable on public databases such as the NCBI), including 395microorganism genomes and a variety of yeast, fungi, plant, andmammalian genomes, the identification of genes encoding the requisite2H3M4OP, p-toluate or terephthalate biosynthetic activity for one ormore genes in related or distant species, including for example,homologues, orthologs, paralogs and nonorthologous gene displacements ofknown genes, and the interchange of genetic alterations betweenorganisms is routine and well known in the art. Accordingly, themetabolic alterations allowing biosynthesis of 2H3M4OP, p-toluate orterephthalate described herein with reference to a particular organismsuch as E. coli can be readily applied to other microorganisms,including prokaryotic and eukaryotic organisms alike. Given theteachings and guidance provided herein, those skilled in the art willknow that a metabolic alteration exemplified in one organism can beapplied equally to other organisms.

In some instances, such as when an alternative 2H3M4OP, p-toluate orterephthalate biosynthetic pathway exists in an unrelated species,2H3M4OP, p-toluate or terephthalate biosynthesis can be conferred ontothe host species by, for example, exogenous expression of a paralog orparalogs from the unrelated species that catalyzes a similar, yetnon-identical metabolic reaction to replace the referenced reaction.Because certain differences among metabolic networks exist betweendifferent organisms, those skilled in the art will understand that theactual gene usage between different organisms may differ. However, giventhe teachings and guidance provided herein, those skilled in the artalso will understand that the teachings and methods of the invention canbe applied to all microbial organisms using the cognate metabolicalterations to those exemplified herein to construct a microbialorganism in a species of interest that will synthesize 2H3M4OP,p-toluate or terephthalate.

Methods for constructing and testing the expression levels of anon-naturally occurring 2H3M4OP, p-toluate or terephthalate producinghost can be performed, for example, by recombinant and detection methodswell known in the art. Such methods can be found described in, forexample, Sambrook et al., Molecular Cloning: A Laboratory Manual, ThirdEd., Cold Spring Harbor Laboratory, New York (2001); and Ausubel et al.,Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore,Md. (1999).

Exogenous nucleic acid sequences involved in a pathway for production of2H3M4OP, p-toluate or terephthalate can be introduced stably ortransiently into a host cell using techniques well known in the artincluding, but not limited to, conjugation, electroporation, chemicaltransformation, transduction, transfection, and ultrasoundtransformation. For exogenous expression in E. coli or other prokaryoticcells, some nucleic acid sequences in the genes or cDNAs of eukaryoticnucleic acids can encode targeting signals such as an N-terminalmitochondrial or other targeting signal, which can be removed beforetransformation into prokaryotic host cells, if desired. For example,removal of a mitochondrial leader sequence led to increased expressionin E. coli (Hoffmeister et al., J. Biol. Chem. 280:4329-4338 (2005)).For exogenous expression in yeast or other eukaryotic cells, genes canbe expressed in the cytosol without the addition of leader sequence, orcan be targeted to mitochondrion or other organelles, or targeted forsecretion, by the addition of a suitable targeting sequence such as amitochondrial targeting or secretion signal suitable for the host cells.Thus, it is understood that appropriate modifications to a nucleic acidsequence to remove or include a targeting sequence can be incorporatedinto an exogenous nucleic acid sequence to impart desirable properties.Furthermore, genes can be subjected to codon optimization withtechniques well known in the art to achieve optimized expression of theproteins.

An expression vector or vectors can be constructed to include one ormore muconate, 2H3M4OP, p-toluate or terephthalate biosynthetic pathwayencoding nucleic acids as exemplified herein operably linked toexpression control sequences functional in the host organism. Anexpression vector or vectors can be constructed to include one or moremuconate biosynthetic pathway encoding nucleic acids as exemplifiedherein operably linked to expression control sequences functional in thehost organism, as disclosed in U.S. Patent Publication No. US2011/0207185 A1. Expression vectors applicable for use in the microbialhost organisms of the invention include, for example, plasmids, phagevectors, viral vectors, episomes and artificial chromosomes, includingvectors and selection sequences or markers operable for stableintegration into a host chromosome. Additionally, the expression vectorscan include one or more selectable marker genes and appropriateexpression control sequences. Selectable marker genes also can beincluded that, for example, provide resistance to antibiotics or toxins,complement auxotrophic deficiencies, or supply critical nutrients not inthe culture media. Expression control sequences can include constitutiveand inducible promoters, transcription enhancers, transcriptionterminators, and the like which are well known in the art. When two ormore exogenous encoding nucleic acids are to be co-expressed, bothnucleic acids can be inserted, for example, into a single expressionvector or in separate expression vectors. For single vector expression,the encoding nucleic acids can be operationally linked to one commonexpression control sequence or linked to different expression controlsequences, such as one inducible promoter and one constitutive promoter.The transformation of exogenous nucleic acid sequences involved in ametabolic or synthetic pathway can be confirmed using methods well knownin the art. Such methods include, for example, nucleic acid analysissuch as Northern blots or polymerase chain reaction (PCR) amplificationof mRNA, or immunoblotting for expression of gene products, or othersuitable analytical methods to test the expression of an introducednucleic acid sequence or its corresponding gene product. It isunderstood by those skilled in the art that the exogenous nucleic acidis expressed in a sufficient amount to produce the desired product, andit is further understood that expression levels can be optimized toobtain sufficient expression using methods well known in the art and asdisclosed herein.

The invention additionally provides a method for producing 2H3M4OP, byculturing the non-naturally occurring microbial organism containing a2H3M4OP pathway under conditions and for a sufficient period of time toproduce 2H3M4OP. Such a microbial organism can have a 2H3M4OP pathwayand include at least one exogenous nucleic acid encoding a 2H3M4OPpathway enzyme expressed in a sufficient amount to produce 2H3M4OP. The2H3M4OP pathway of the microbial organism can include a pathway selectedfrom: (1) 1A, 1B, 1C, 1D, 1E and 1F; (2) 2A, 2B and 2C; and (3) 2D, 2Eand 2C, wherein 1A is an erythrose-4-phosphate dehydrogenase, wherein 1Bis a 4-phosphoerythronate dehydrogenase, wherein 1C is a2-acetyl-2,3-dihydroxy-4-phosphobutanoate synthase, wherein 1D is a2-acetyl-2,3-dihydroxy-4-phosphobutanoate reductoisomerase, wherein 1Eis a 2,3,4-trihydroxy-3-methyl-5-phosphopentanoate dehydratase, wherein1F is a 4-hydroxy-3-methyl-2-oxo-5-phosphopentanoate decarboxylase,wherein 2A is a 4,5-dihydroxy-2-oxopentanoate methyltransferase, wherein2B is a 4,5-dihydroxy-3-methyl-2-oxopentanoate kinase, wherein 2C is a4-hydroxy-3-methyl-2-oxo-5-phosphopentanoate decarboxylase, wherein 2Dis a 4,5-dihydroxy-2-oxopentanoate kinase, wherein 2E is a4-hydroxy-2-oxo-5-phosphopentanoate methyltransferase (see FIGS. 1 and 2and Example I). Additionally, in some aspects, the microbial organismcultured in the methods of the invention can include two, three, four,five or six exogenous nucleic acids, wherein each exogenous nucleic acidencodes a 2H3M4OP pathway enzyme as described herein. In some aspects,the invention provides a method for producing 2H3M4OP by culturing amicrobial organism of the invention having exogenous nucleic acidsencoding each of the 2H3M4OP pathway enzymes of at least one of the2H3M4OP pathways selected from (1)-(3), as described above.

In some aspects, the invention provides the 2H3M4OP pathway of themicrobial organism cultured in the invention methods includes a2-C-methyl-D-erythritol-4-phosphate dehydratase (see Example II and FIG.3, step C). The non-naturally occurring microbial organism having a2H3M4OP pathway can further have a 1-deoxyxylulose-5-phosphate synthaseor a 1-deoxy-D-xylulose-5-phosphate reductoisomerase (see Example II andFIG. 3, steps A and B). Thus, a 2H3M4OP pathway can include a2-C-methyl-D-erythritol-4-phosphate dehydratase, a1-deoxyxylulose-5-phosphate synthase and a1-deoxy-D-xylulose-5-phosphate reductoisomerase.

The invention also provides a method for producing p-toluate byculturing the non-naturally occurring microbial organism containing a2H3M4OP pathway and/or a p-toluate pathway under conditions and for asufficient period of time to produce p-toluate. Such a microbialorganism can include at least one exogenous nucleic acid encoding ap-toluate pathway enzyme expressed in a sufficient amount to producep-toluate. In some aspects, the p-toluate pathway includes 4A, 4B, 4C,4D, 4E, 4F, 4G and/or 4H, wherein 4A is a2-dehydro-3-deoxyphosphoheptonate synthase; wherein 4B is a3-dehydroquinate synthase; wherein 4C is a 3-dehydroquinate dehydratase;wherein 4D is a shikimate dehydrogenase; wherein 4E is a shikimatekinase; wherein 4F is a 3-phosphoshikimate-2-carboxyvinyltransferase;wherein 4G is a chorismate synthase and wherein 4H is a chorismate lyase(see Example III and FIG. 4, steps A-H). A non-naturally occurringmicrobial organism having a p-toluate pathway can further include a2H3M4OP pathway as described herein (see Examples I and II and FIGS.1-3). For example, a 2H3M4OP pathway can include a pathway selectedfrom: (1) 1A, 1B, 1C, 1D, 1E and 1F; (2) 2A, 2B and 2C; and (3) 2D, 2Eand 2C, wherein 1A is an erythrose-4-phosphate dehydrogenase, wherein 1Bis a 4-phosphoerythronate dehydrogenase, wherein 1C is a2-acetyl-2,3-dihydroxy-4-phosphobutanoate synthase, wherein 1D is a2-acetyl-2,3-dihydroxy-4-phosphobutanoate reductoisomerase, wherein 1Eis a 2,3,4-trihydroxy-3-methyl-5-phosphopentanoate dehydratase, wherein1F is a 4-hydroxy-3-methyl-2-oxo-5-phosphopentanoate decarboxylase,wherein 2A is a 4,5-dihydroxy-2-oxopentanoate methyltransferase, wherein2B is a 4,5-dihydroxy-3-methyl-2-oxopentanoate kinase, wherein 2C is a4-hydroxy-3-methyl-2-oxo-5-phosphopentanoate decarboxylase, wherein 2Dis a 4,5-dihydroxy-2-oxopentanoate kinase, wherein 2E is a4-hydroxy-2-oxo-5-phosphopentanoate methyltransferase (see FIGS. 1 and 2and Example I). Alternatively, a 2H3M4OP pathway can include a2-C-methyl-D-erythritol-4-phosphate dehydratase, a1-deoxyxylulose-5-phosphate synthase and/or a1-deoxy-D-xylulose-5-phosphate reductoisomerase (see FIG. 3 and ExampleII).

In some aspects, the microbial organism cultured in the inventionmethods include two, three, four, five, six, seven or eight exogenousnucleic acids, wherein each nucleic acid encodes a p-toluate pathwayenzyme. Additionally, in some aspects, the invention provides that themicrobial organism cultured in the invention methods includes exogenousnucleic acids encoding each of the enzymes of the p-toluate pathwaydisclosed herein.

The invention also provides a method for producing terephthalate byculturing the non-naturally occurring microbial organism containing a2H3M4OP pathway, a p-toluate pathway and/or a terephthalate pathwayunder conditions and for a sufficient period of time to produceterephthalate. Such a microbial organism can include at least oneexogenous nucleic acid encoding a terephthalate pathway enzyme expressedin a sufficient amount to produce terephthalate. In some aspects of theinvention, the terephthalate pathway can include 5A, 5B and 5C, wherein5A is a p-toluate methyl-monooxygenase reductase, wherein 5B is a4-carboxybenzyl alcohol dehydrogenase and wherein 5C is a4-carboxybenzyl aldehyde dehydrogenase (see Example IV and FIG. 5). Suchan organism containing a terephthalate pathway can additionally includea p-toluate pathway, wherein the p-toluate pathway includes 4A, 4B, 4C,4D, 4E, 4F, 4G and/or 4H, wherein 4A is a2-dehydro-3-deoxyphosphoheptonate synthase; wherein 4B is a3-dehydroquinate synthase; wherein 4C is a 3-dehydroquinate dehydratase;wherein 4D is a shikimate dehydrogenase; wherein 4E is a shikimatekinase; wherein 4F is a 3-phosphoshikimate-2-carboxyvinyltransferase;wherein 4G is a chorismate synthase and wherein 4H is a chorismate lyase(see Examples III and IV and FIGS. 4 and 5). Such a non-naturallyoccurring microbial organism having a terephthalate pathway and ap-toluate pathway can further include a 2H3M4OP pathway as describedherein (see Examples I and II and FIGS. 1-3). For example, a 2H3M4OPpathway can include a pathway selected from: (1) 1A, 1B, 1C, 1D, 1E and1F; (2) 2A, 2B and 2C; and (3) 2D, 2E and 2C, wherein 1A is anerythrose-4-phosphate dehydrogenase, wherein 1B is a4-phosphoerythronate dehydrogenase, wherein 1C is a2-acetyl-2,3-dihydroxy-4-phosphobutanoate synthase, wherein 1D is a2-acetyl-2,3-dihydroxy-4-phosphobutanoate reductoisomerase, wherein 1Eis a 2,3,4-trihydroxy-3-methyl-5-phosphopentanoate dehydratase, wherein1F is a 4-hydroxy-3-methyl-2-oxo-5-phosphopentanoate decarboxylase,wherein 2A is a 4,5-dihydroxy-2-oxopentanoate methyltransferase, wherein2B is a 4,5-dihydroxy-3-methyl-2-oxopentanoate kinase, wherein 2C is a4-hydroxy-3-methyl-2-oxo-5-phosphopentanoate decarboxylase, wherein 2Dis a 4,5-dihydroxy-2-oxopentanoate kinase, wherein 2E is a4-hydroxy-2-oxo-5-phosphopentanoate methyltransferase (see FIGS. 1 and 2and Example I). Alternatively, a 2H3M4OP pathway can include a2-C-methyl-D-erythritol-4-phosphate dehydratase, a1-deoxyxylulose-5-phosphate synthase and/or a1-deoxy-D-xylulose-5-phosphate reductoisomerase (see FIG. 3 and ExampleII).

In some aspects, the microbial organism cultured in the inventionmethods includes two or three exogenous nucleic acids, wherein eachnucleic acid encodes a terephthalate pathway enzyme. Additionally, insome aspects, the invention provides that the microbial organismcultured in the invention methods includes exogenous nucleic acidsencoding each of the enzymes of the terephthalate pathway disclosedherein.

Suitable purification and/or assays to test for the production of2H3M4OP, p-toluate or terephthalate can be performed using well knownmethods. Suitable replicates such as triplicate cultures can be grownfor each engineered strain to be tested. For example, product andbyproduct formation in the engineered production host can be monitored.The final product and intermediates, and other organic compounds, can beanalyzed by methods such as HPLC (High Performance LiquidChromatography), GC-MS (Gas Chromatography-Mass Spectroscopy) and LC-MS(Liquid Chromatography-Mass Spectroscopy) or other suitable analyticalmethods using routine procedures well known in the art. The release ofproduct in the fermentation broth can also be tested with the culturesupernatant. Byproducts and residual glucose can be quantified by HPLCusing, for example, a refractive index detector for glucose andalcohols, and a UV detector for organic acids (Lin et al., Biotechnol.Bioeng. 90:775-779 (2005)), or other suitable assay and detectionmethods well known in the art. The individual enzyme or proteinactivities from the exogenous DNA sequences can also be assayed usingmethods well known in the art. For example, p-toluatemethyl-monooxygenase activity can be assayed by incubating purifiedenzyme with NADH, FeSO₄ and the p-toluate substrate in a water bath,stopping the reaction by precipitation of the proteins, and analysis ofthe products in the supernatant by HPLC (Locher et al., J. Bacteriol.173:3741-3748 (1991)).

The 2H3M4OP, p-toluate or terephthalate can be separated from othercomponents in the culture using a variety of methods well known in theart. Such separation methods include, for example, extraction proceduresas well as methods that include continuous liquid-liquid extraction,pervaporation, membrane filtration, membrane separation, reverseosmosis, electrodialysis, distillation, crystallization, centrifugation,extractive filtration, ion exchange chromatography, size exclusionchromatography, adsorption chromatography, and ultrafiltration. All ofthe above methods are well known in the art.

Any of the non-naturally occurring microbial organisms described hereincan be cultured to produce and/or secrete the biosynthetic products ofthe invention. For example, the muconate, 2H3M4OP, p-toluate orterephthalate producers can be cultured for the biosynthetic productionof muconate, 2H3M4OP, p-toluate or terephthalate. Accordingly, in someembodiments, the invention provides culture medium having muconate,2H3M4OP, p-toluate or terephthalate or a muconate, 2H3M4OP, p-toluate orterephthalate pathway intermediate described herein. In some aspects,the culture mediums can also be separated from the non-naturallyoccurring microbial organisms of the invention that produced themuconate, 2H3M4OP, p-toluate or terephthalate or muconate, 2H3M4OP,p-toluate or terephthalate pathway intermediate. Methods for separatinga microbial organism from culture medium are well known in the art.Exemplary methods include filtration, flocculation, precipitation,centrifugation, sedimentation, and the like.

For the production of 2H3M4OP, p-toluate or terephthalate, therecombinant strains are cultured in a medium with carbon source andother essential nutrients. It is sometimes desirable and can be highlydesirable to maintain anaerobic conditions in the fermenter to reducethe cost of the overall process. Such conditions can be obtained, forexample, by first sparging the medium with nitrogen and then sealing theflasks with a septum and crimp-cap. For strains where growth is notobserved anaerobically, microaerobic or substantially anaerobicconditions can be applied by perforating the septum with a small holefor limited aeration. Exemplary anaerobic conditions have been describedpreviously and are well-known in the art. Exemplary aerobic andanaerobic conditions are described, for example, in United Statepublication 2009/0047719, filed Aug. 10, 2007. Exemplary cell growthprocedures and fermentations used in the production of a compound ofinterest, such as, for example, muconate, 2H3M4OP, p-toluate orterephthalate, include, batch fermentation, fed-batch fermentation withbatch separation; fed-batch fermentation with continuous separation, andcontinuous fermentation with continuous separation. All of theseprocesses are well known in the art. Depending on the non-naturallyoccurring microbial organism's design, the fermentations can be carriedout under aerobic or anaerobic conditions. In certain embodiments, thetemperature of the cultures are kept between about 30 and about 45° C.,including 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, and 44° C.

In batch fermentation, a tank fermenter (or bioreactor) is filled withthe prepared media to support growth. The temperature and pH formicrobial fermentation is properly adjusted, and any additionalsupplements are added. An inoculum of a producing non-naturallyoccurring microbial organism is added to the fermenter. In batchfermentation the fermentation will generally run for a fixed period andthen the products from the fermentation are isolated. The process can berepeated in batch runs.

In fed-batch fermentation fresh media is continuously or periodicallyadded to the fermentation bioreactor. Fixed-volume fed-batchfermentation is a type of fed-batch fermentation in which a carbonsource is fed without diluting the culture. The culture volume can alsobe maintained nearly constant by feeding the growth carbon source as aconcentrated liquid or gas. In another type of fixed-volume fed-batchculture, sometimes called a cyclic fed-batch culture, a portion of theculture is periodically withdrawn and used as the starting point for afurther fed-batch process. Once the fermentation reaches a certainstage, the culture is removed and the biomass is diluted to the originalvolume with sterile water or medium containing the carbon feedsubstrate. The dilution decreases the biomass concentration and resultsin an increase in the specific growth rate. Subsequently, as feedingcontinues, the growth rate will decline gradually as biomass increasesand approaches the maximum sustainable in the vessel once more, at whichpoint the culture can be diluted again. Alternatively, a fed-batchfermentation can be variable volume. In variable-volume mode the volumeof the fermentation broth changes with the fermentation time as nutrientand media are continually added to the culture without removal of aportion of the fermentation broth.

In a continuous fermentation, fresh media is generally continually addedwith continuous separation of spent medium, which can include theproduct of interest, such as, for example, muconate, 2H3M4OP, p-toluateor terephthalate, when the product is secreted. One feature of thecontinuous culture is that a time-independent steady-state can beobtained which enables one to determine the relations between microbialbehavior and the environmental conditions. Achieving this steady-stateis accomplished by means of a chemostat, or similar bioreactor. Achemostat allows for the continual addition of fresh medium whileculture liquid is continuously removed to keep the culture volumeconstant. By altering the rate at which medium is added to thechemostat, the growth rate of the non-naturally occurring microbialorganism can be controlled.

The continuous and/or near-continuous production of a compound ofinterest, such as, for example, muconate, 2H3M4OP, p-toluate orterephthalate, can include culturing a compound-producing non-naturallyoccurring microbial organism in sufficient nutrients and medium tosustain and/or nearly sustain growth in an exponential phase. Continuousculture under such conditions can include, for example, 1 day, 2, 3, 4,5, 6 or 7 days or more. Additionally, continuous culture can include 1week, 2, 3, 4 or 5 or more weeks and up to several months.Alternatively, organisms that produce a compound of interest can becultured for hours, if suitable for a particular application. It is tobe understood that the continuous and/or near-continuous cultureconditions also can include all time intervals in between theseexemplary periods. It is further understood that the time of culturingthe compound-producing non-naturally occurring microbial organism is fora sufficient period of time to produce a sufficient amount of productfor a desired purpose.

In certain embodiments, the culture can be conducted under aerobicconditions. An oxygen feed to the culture can be controlled. Oxygen canbe supplied as air, enriched oxygen, pure oxygen or any combinationthereof. Methods of monitoring oxygen concentration are known in theart. Oxygen can be delivered at a certain feed rate or can be deliveredon demand by measuring the dissolved oxygen content of the culture andfeeding accordingly with the intention of maintaining a constantdissolved oxygen content.

Fermentations can be performed under anaerobic conditions. For example,as explained above, the culture can be rendered substantially free ofoxygen by first sparging the medium with nitrogen and then sealingculture vessel (e.g., flasks can be sealed with a septum and crimp-cap).Microaerobic conditions also can be utilized by providing a small holefor limited aeration. On a commercial scale, microaerobic conditions areachieved by sparging a fermentor with air or oxygen as in the aerobiccase, but at a much lower rate and with tightly controlled agitation.

If desired, the pH of the medium can be maintained at a desired pH, inparticular neutral pH, such as a pH of around 7 by addition of a base,such as NaOH or other bases, or acid, as needed to maintain the culturemedium at a desirable pH.

The growth rate can be determined by measuring optical density using aspectrophotometer (600 nm), and the glucose uptake rate by monitoringcarbon source depletion over time.

The growth medium can include, for example, any carbohydrate sourcewhich can supply a source of carbon to the non-naturally occurringmicroorganism. Such sources include, for example: sugars such asglucose, xylose, arabinose, galactose, mannose, fructose, sucrose andstarch; glycerol; carbon dioxide; formate; methane; methanol; or acarbon source generated from electrochemical conversion of carbondioxide, such as formate or methanol, alone as the sole source of carbonor in combination with other carbon sources described herein or known inthe art. Other sources of carbohydrate include, for example, renewablefeedstocks and biomass. Exemplary types of biomasses that can be used asfeedstocks in the methods of the invention include cellulosic biomass,hemicellulosic biomass and lignin feedstocks or portions of feedstocks.Such biomass feedstocks contain, for example, carbohydrate substratesuseful as carbon sources such as glucose, xylose, arabinose, galactose,mannose, fructose and starch. Given the teachings and guidance providedherein, those skilled in the art will understand that renewablefeedstocks and biomass other than those exemplified above also can beused for culturing the microbial organisms of the invention for theproduction of 2H3M4OP, p-toluate or terephthalate.

In addition to renewable feedstocks such as those exemplified above, the2H3M4OP, p-toluate or terephthalate microbial organisms of the inventionalso can be modified for growth on syngas as its source of carbon. Inthis specific embodiment, one or more proteins or enzymes are expressedin the 2H3M4OP, p-toluate or terephthalate producing organisms toprovide a metabolic pathway for utilization of syngas or other gaseouscarbon source.

Synthesis gas, also known as syngas or producer gas, is the majorproduct of gasification of coal and of carbonaceous materials such asbiomass materials, including agricultural crops and residues. Syngas isa mixture primarily of H₂ and CO and can be obtained from thegasification of any organic feedstock, including but not limited tocoal, coal oil, natural gas, biomass, and waste organic matter.Gasification is generally carried out under a high fuel to oxygen ratio.Although largely H₂ and CO, syngas can also include CO₂ and other gasesin smaller quantities. Thus, synthesis gas provides a cost effectivesource of gaseous carbon such as CO and, additionally, CO₂.

The Wood-Ljungdahl pathway catalyzes the conversion of CO and H₂ toacetyl-CoA and other products such as acetate. Organisms capable ofutilizing CO and syngas also generally have the capability of utilizingCO₂ and CO₂/H₂ mixtures through the same basic set of enzymes andtransformations encompassed by the Wood-Ljungdahl pathway. H₂-dependentconversion of CO₂ to acetate by microorganisms was recognized longbefore it was revealed that CO also could be used by the same organismsand that the same pathways were involved. Many acetogens have been shownto grow in the presence of CO₂ and produce compounds such as acetate aslong as hydrogen is present to supply the necessary reducing equivalents(see for example, Drake, Acetogenesis, pp. 3-60 Chapman and Hall, NewYork, (1994)). This can be summarized by the following equation:

2CO₂+4H₂ +nADP+nPi→CH₃COOH+2H₂O+nATP

Hence, non-naturally occurring microorganisms possessing theWood-Ljungdahl pathway can utilize CO₂ and H₂ mixtures as well for theproduction of acetyl-CoA and other desired products.

The Wood-Ljungdahl pathway is well known in the art and consists of 12reactions which can be separated into two branches: (1) methyl branchand (2) carbonyl branch. The methyl branch converts syngas tomethyl-tetrahydrofolate (methyl-THF) whereas the carbonyl branchconverts methyl-THF to acetyl-CoA. The reactions in the methyl branchare catalyzed in order by the following enzymes or proteins: ferredoxinoxidoreductase, formate dehydrogenase, formyltetrahydrofolatesynthetase, methenyltetrahydrofolate cyclodehydratase,methylenetetrahydrofolate dehydrogenase and methylenetetrahydrofolatereductase. The reactions in the carbonyl branch are catalyzed in orderby the following enzymes or proteins: methyltetrahydrofolate:corrinoidprotein methyltransferase (for example, AcsE), corrinoid iron-sulfurprotein, nickel-protein assembly protein (for example, AcsF),ferredoxin, acetyl-CoA synthase, carbon monoxide dehydrogenase andnickel-protein assembly protein (for example, CooC). Following theteachings and guidance provided herein for introducing a sufficientnumber of encoding nucleic acids to generate a 2H3M4OP, p-toluate orterephthalate pathway, those skilled in the art will understand that thesame engineering design also can be performed with respect tointroducing at least the nucleic acids encoding the Wood-Ljungdahlenzymes or proteins absent in the host organism. Therefore, introductionof one or more encoding nucleic acids into the microbial organisms ofthe invention such that the modified organism contains the completeWood-Ljungdahl pathway will confer syngas utilization ability.

Additionally, the reductive (reverse) tricarboxylic acid cycle coupledwith carbon monoxide dehydrogenase and/or hydrogenase activities canalso be used for the conversion of CO, CO₂ and/or H₂ to acetyl-CoA andother products such as acetate. Organisms capable of fixing carbon viathe reductive TCA pathway can utilize one or more of the followingenzymes: ATP citrate-lyase, citrate lyase, aconitase, isocitratedehydrogenase, alpha-ketoglutarate:ferredoxin oxidoreductase,succinyl-CoA synthetase, succinyl-CoA transferase, fumarate reductase,fumarase, malate dehydrogenase, NAD(P)H:ferredoxin oxidoreductase,carbon monoxide dehydrogenase, and hydrogenase. Specifically, thereducing equivalents extracted from CO and/or H₂ by carbon monoxidedehydrogenase and hydrogenase are utilized to fix CO₂ via the reductiveTCA cycle into acetyl-CoA or acetate. Acetate can be converted toacetyl-CoA by enzymes such as acetyl-CoA transferase, acetatekinase/phosphotransacetylase, and acetyl-CoA synthetase. Acetyl-CoA canbe converted to the 2H3M4OP, p-toluate or terephthalate precursors,glyceraldehyde-3-phosphate, phosphoenolpyruvate, and pyruvate, bypyruvate:ferredoxin oxidoreductase and the enzymes of gluconeogenesis.Following the teachings and guidance provided herein for introducing asufficient number of encoding nucleic acids to generate a 2H3M4OP,p-toluate or terephthalate pathway, those skilled in the art willunderstand that the same engineering design also can be performed withrespect to introducing at least the nucleic acids encoding the reductiveTCA pathway enzymes or proteins absent in the host organism. Therefore,introduction of one or more encoding nucleic acids into the microbialorganisms of the invention such that the modified organism contains areductive TCA pathway can confer syngas utilization ability.

Accordingly, given the teachings and guidance provided herein, thoseskilled in the art will understand that a non-naturally occurringmicrobial organism can be produced that secretes the biosynthesizedcompounds of the invention when grown on a carbon source such as acarbohydrate. Such compounds include, for example, 2H3M4OP, p-toluate orterephthalate and any of the intermediate metabolites in the 2H3M4OP,p-toluate or terephthalate pathway. All that is required is to engineerin one or more of the required enzyme or protein activities to achievebiosynthesis of the desired compound or intermediate including, forexample, inclusion of some or all of the 2H3M4OP, p-toluate orterephthalate biosynthetic pathways. Accordingly, the invention providesa non-naturally occurring microbial organism that produces and/orsecretes 2H3M4OP, p-toluate or terephthalate when grown on acarbohydrate or other carbon source and produces and/or secretes any ofthe intermediate metabolites shown in the 2H3M4OP, p-toluate orterephthalate pathway when grown on a carbohydrate or other carbonsource. The 2H3M4OP, p-toluate or terephthalate producing microbialorganisms of the invention can initiate synthesis from an intermediate.For example, a 2H3M4OP pathway intermediate can be 4-phosphoerythronate,2-oxo-3-hydroxy-4-phosphobutanoate, 2-acetyl-2,3-phosphodutanoate,2-acetyl-2,3-dihydroxy-4-phosphobutanoate,2,3,4-trihydroxy-3-methyl-5-phosphopentanoate,4-hydroxy-3-methyl-2-oxo-5-phosphopentanoate,4,5-dihydroxy-3-methyl-2-oxopentanoate,4-hydroxy-2-oxo-5-phosphopentanoate, 1-deoxy-D-xylulose-5-phosphate orC-methyl-D-erythritol-4-phosphate (see Examples I and II and FIGS. 1-3).A p-toluate pathway intermediate can be, for example,2,4-dihydroxy-5-methyl-6-[(phosphonooxy)methyl]oxane-2-carboxylate,1,3-dihydroxy-4-methyl-5-oxocyclohexane-1-carboxylate,5-hydroxy-4-methyl-3-oxocyclohex-1-ene-1-carboxylate,3,5-dihydroxy-4-methylcyclohex-1-ene-1-carboxylate,5-hydroxy-4-methyl-3-(phosphonooxy)cyclohex-1-ene-1-carboxylate,5-[(1-carboxyeth-1-en-1-yl)oxy]-4-methyl-3-(phosphonooxy)cyclohex-1-ene-1-carboxylate,or3-[(1-carboxyeth-1-en-1-yl)oxy]-4-methylcyclohexa-1,5-diene-1-carboxylate(see Example II and FIG. 2). A terephthalate intermediate can be, forexample, 4-carboxybenzyl alcohol or 4-carboxybenzaldehyde (see ExampleIII and FIG. 3).

The non-naturally occurring microbial organisms of the invention areconstructed using methods well known in the art as exemplified herein toexogenously express at least one nucleic acid encoding a 2H3M4OP,p-toluate or terephthalate pathway enzyme or protein in sufficientamounts to produce 2H3M4OP, p-toluate or terephthalate. It is understoodthat the microbial organisms of the invention are cultured underconditions sufficient to produce 2H3M4OP, p-toluate or terephthalate.Following the teachings and guidance provided herein, the non-naturallyoccurring microbial organisms of the invention can achieve biosynthesisof 2H3M4OP, p-toluate or terephthalate resulting in intracellularconcentrations between about 0.1-200 mM or more. Generally, theintracellular concentration of 2H3M4OP, p-toluate or terephthalate isbetween about 3-150 mM, particularly between about 5-125 mM and moreparticularly between about 8-100 mM, including about 10 mM, 20 mM, 50mM, 80 mM, or more. Intracellular concentrations between and above eachof these exemplary ranges also can be achieved from the non-naturallyoccurring microbial organisms of the invention.

In some embodiments, culture conditions include anaerobic orsubstantially anaerobic growth or maintenance conditions. Exemplaryanaerobic conditions have been described previously and are well knownin the art. Exemplary anaerobic conditions for fermentation processesare described herein and are described, for example, in U.S. publication2009/0047719, filed Aug. 10, 2007. Any of these conditions can beemployed with the non-naturally occurring microbial organisms as well asother anaerobic conditions well known in the art. Under such anaerobicor substantially anaerobic conditions, the 2H3M4OP, p-toluate orterephthalate producers can synthesize 2H3M4OP, p-toluate orterephthalate at intracellular concentrations of 5-10 mM or more as wellas all other concentrations exemplified herein. It is understood that,even though the above description refers to intracellularconcentrations, 2H3M4OP, p-toluate or terephthalate producing microbialorganisms can produce 2H3M4OP, p-toluate or terephthalateintracellularly and/or secrete the product into the culture medium.

Exemplary fermentation processes include, but are not limited to,fed-batch fermentation and batch separation; fed-batch fermentation andcontinuous separation; and continuous fermentation and continuousseparation. In an exemplary batch fermentation protocol, the productionorganism is grown in a suitably sized bioreactor sparged with anappropriate gas. Under anaerobic conditions, the culture is sparged withan inert gas or combination of gases, for example, nitrogen, N₂/CO₂mixture, argon, helium, and the like. As the cells grow and utilize thecarbon source, additional carbon source(s) and/or other nutrients arefed into the bioreactor at a rate approximately balancing consumption ofthe carbon source and/or nutrients. The temperature of the bioreactor ismaintained at a desired temperature, generally in the range of 22-37degrees C., but the temperature can be maintained at a higher or lowertemperature depending on the growth characteristics of the productionorganism and/or desired conditions for the fermentation process. Growthcontinues for a desired period of time to achieve desiredcharacteristics of the culture in the fermenter, for example, celldensity, product concentration, and the like. In a batch fermentationprocess, the time period for the fermentation is generally in the rangeof several hours to several days, for example, 8 to 24 hours, or 1, 2,3, 4 or 5 days, or up to a week, depending on the desired cultureconditions. The pH can be controlled or not, as desired, in which case aculture in which pH is not controlled will typically decrease to pH 3-6by the end of the run. Upon completion of the cultivation period, thefermenter contents can be passed through a cell separation unit, forexample, a centrifuge, filtration unit, and the like, to remove cellsand cell debris. In the case where the desired product is expressedintracellularly, the cells can be lysed or disrupted enzymatically orchemically prior to or after separation of cells from the fermentationbroth, as desired, in order to release additional product. Thefermentation broth can be transferred to a product separations unit.Isolation of product occurs by standard separations procedures employedin the art to separate a desired product from dilute aqueous solutions.Such methods include, but are not limited to, liquid-liquid extractionusing a water immiscible organic solvent (e.g, toluene or other suitablesolvents, including but not limited to diethyl ether, ethyl acetate,tetrahydrofuran (THF), methylene chloride, chloroform, benzene, pentane,hexane, heptane, petroleum ether, methyl tertiary butyl ether (MTBE),dioxane, dimethylformamide (DMF), dimethyl sulfoxide (DMSO), and thelike) to provide an organic solution of the product, if appropriate,standard distillation methods, and the like, depending on the chemicalcharacteristics of the product of the fermentation process.

In an exemplary fully continuous fermentation protocol, the productionorganism is generally first grown up in batch mode in order to achieve adesired cell density. When the carbon source and/or other nutrients areexhausted, feed medium of the same composition is supplied continuouslyat a desired rate, and fermentation liquid is withdrawn at the samerate. Under such conditions, the product concentration in the bioreactorgenerally remains constant, as well as the cell density. The temperatureof the fermenter is maintained at a desired temperature, as discussedabove. During the continuous fermentation phase, it is generallydesirable to maintain a suitable pH range for optimized production. ThepH can be monitored and maintained using routine methods, including theaddition of suitable acids or bases to maintain a desired pH range. Thebioreactor is operated continuously for extended periods of time,generally at least one week to several weeks and up to one month, orlonger, as appropriate and desired. The fermentation liquid and/orculture is monitored periodically, including sampling up to every day,as desired, to assure consistency of product concentration and/or celldensity. In continuous mode, fermenter contents are constantly removedas new feed medium is supplied. The exit stream, containing cells,medium, and product, are generally subjected to a continuous productseparations procedure, with or without removing cells and cell debris,as desired. Continuous separations methods employed in the art can beused to separate the product from dilute aqueous solutions, includingbut not limited to continuous liquid-liquid extraction using a waterimmiscible organic solvent (e.g., toluene or other suitable solvents,including but not limited to diethyl ether, ethyl acetate,tetrahydrofuran (THF), methylene chloride, chloroform, benzene, pentane,hexane, heptane, petroleum ether, methyl tertiary butyl ether (MTBE),dioxane, dimethylformamide (DMF), dimethyl sulfoxide (DMSO), and thelike), standard continuous distillation methods, and the like, or othermethods well known in the art.

In addition to the culturing and fermentation conditions disclosedherein, growth conditions for achieving biosynthesis of 2H3M4OP,p-toluate or terephthalate can include the addition of an osmoprotectantto the culturing conditions, as described, for example, in U.S. PatentPublication No. US 2011/0207185 A1.

In addition to the culturing and fermentation conditions disclosedherein, growth condition for achieving biosynthesis of 2H3M4OP,p-toluate or terephthalate can include the addition of an osmoprotectantto the culturing conditions. In certain embodiments, the non-naturallyoccurring microbial organisms of the invention can be sustained,cultured or fermented as described herein in the presence of anosmoprotectant. Briefly, an osmoprotectant refers to a compound thatacts as an osmolyte and helps a microbial organism as described hereinsurvive osmotic stress. Osmoprotectants include, but are not limited to,betaines, amino acids, and the sugar trehalose. Non-limiting examples ofsuch are glycine betaine, praline betaine, dimethylthetin,dimethylslfonioproprionate, 3-dimethylsulfonio-2-methylproprionate,pipecolic acid, dimethylsulfonioacetate, choline, L-carnitine andectoine. In one aspect, the osmoprotectant is glycine betaine. It isunderstood to one of ordinary skill in the art that the amount and typeof osmoprotectant suitable for protecting a microbial organism describedherein from osmotic stress will depend on the microbial organism used.The amount of osmoprotectant in the culturing conditions can be, forexample, no more than about 0.1 mM, no more than about 0.5 mM, no morethan about 1.0 mM, no more than about 1.5 mM, no more than about 2.0 mM,no more than about 2.5 mM, no more than about 3.0 mM, no more than about5.0 mM, no more than about 7.0 mM, no more than about 10 mM, no morethan about 50 mM, no more than about 100 mM or no more than about 500mM.

In some embodiments, the carbon feedstock and other cellular uptakesources such as phosphate, ammonia, sulfate, chloride and other halogenscan be chosen to alter the isotopic distribution of the atoms present in2H3M4OP, p-toluate or terephthalate or any 2H3M4OP, p-toluate orterephthalate pathway intermediate. The various carbon feedstock andother uptake sources enumerated above will be referred to herein,collectively, as “uptake sources.” Uptake sources can provide isotopicenrichment for any atom present in the product 2H3M4OP, p-toluate orterephthalate or 2H3M4OP, p-toluate or terephthalate pathwayintermediate, or for side products generated in reactions diverging awayfrom a 2H3M4OP, p-toluate or terephthalate pathway. Isotopic enrichmentcan be achieved for any target atom including, for example, carbon,hydrogen, oxygen, nitrogen, sulfur, phosphorus, chloride or otherhalogens.

In some embodiments, the uptake sources can be selected to alter thecarbon-12, carbon-13, and carbon-14 ratios. In some embodiments, theuptake sources can be selected to alter the oxygen-16, oxygen-17, andoxygen-18 ratios. In some embodiments, the uptake sources can beselected to alter the hydrogen, deuterium, and tritium ratios. In someembodiments, the uptake sources can be selected to alter the nitrogen-14and nitrogen-15 ratios. In some embodiments, the uptake sources can beselected to alter the sulfur-32, sulfur-33, sulfur-34, and sulfur-35ratios. In some embodiments, the uptake sources can be selected to alterthe phosphorus-31, phosphorus-32, and phosphorus-33 ratios. In someembodiments, the uptake sources can be selected to alter thechlorine-35, chlorine-36, and chlorine-37 ratios.

In some embodiments, the isotopic ratio of a target atom can be variedto a desired ratio by selecting one or more uptake sources. An uptakesource can be derived from a natural source, as found in nature, or froma man-made source, and one skilled in the art can select a naturalsource, a man-made source, or a combination thereof, to achieve adesired isotopic ratio of a target atom. An example of a man-made uptakesource includes, for example, an uptake source that is at leastpartially derived from a chemical synthetic reaction. Such isotopicallyenriched uptake sources can be purchased commercially or prepared in thelaboratory and/or optionally mixed with a natural source of the uptakesource to achieve a desired isotopic ratio. In some embodiments, atarget atom isotopic ratio of an uptake source can be achieved byselecting a desired origin of the uptake source as found in nature. Forexample, as discussed herein, a natural source can be derived from orsynthesized by a biological organism or a source such as petroleum-basedproducts or the atmosphere. In some such embodiments, a source ofcarbon, for example, can be selected from a fossil fuel-derived carbonsource, which can be relatively depleted of carbon-14, or anenvironmental or atmospheric carbon source, such as CO₂, which canpossess a larger amount of carbon-14 than its petroleum-derivedcounterpart.

The unstable carbon isotope carbon-14 or radiocarbon makes up forroughly 1 in 10¹² carbon atoms in the earth's atmosphere and has ahalf-life of about 5700 years. The stock of carbon is replenished in theupper atmosphere by a nuclear reaction involving cosmic rays andordinary nitrogen (¹⁴N). Fossil fuels contain no carbon-14, as itdecayed long ago. Burning of fossil fuels lowers the atmosphericcarbon-14 fraction, the so-called “Suess effect”.

Methods of determining the isotopic ratios of atoms in a compound arewell known to those skilled in the art. Isotopic enrichment is readilyassessed by mass spectrometry using techniques known in the art such asaccelerated mass spectrometry (AMS), Stable Isotope Ratio MassSpectrometry (SIRMS) and Site-Specific Natural Isotopic Fractionation byNuclear Magnetic Resonance (SNIF-NMR). Such mass spectral techniques canbe integrated with separation techniques such as liquid chromatography(LC), high performance liquid chromatography (HPLC) and/or gaschromatography, and the like.

In the case of carbon, ASTM D6866 was developed in the United States asa standardized analytical method for determining the bio-based contentof solid, liquid, and gaseous samples using radiocarbon dating by theAmerican Society for Testing and Materials (ASTM) International. Thestandard is based on the use of radiocarbon dating for the determinationof a product's bio-derived content. ASTM D6866 was first published in2004, and the current active version of the standard is ASTM D6866-11(effective Apr. 1, 2011). Radiocarbon dating techniques are well knownto those skilled in the art, including those described herein.

The bio-based content of a compound is estimated by the ratio ofcarbon-14 (¹⁴C) to carbon-12 (¹²C). Specifically, the Fraction Modern(Fm) is computed from the expression: Fm=(S−B)/(M−B), where B, S and Mrepresent the ¹⁴C/¹²C ratios of the blank, the sample and the modernreference, respectively. Fraction Modern is a measurement of thedeviation of the ¹⁴C/¹²C ratio of a sample from “Modern.” Modern isdefined as 95% of the radiocarbon concentration (in AD 1950) of NationalBureau of Standards (NBS) Oxalic Acid I (i.e., standard referencematerials (SRM) 4990b) normalized to δ¹³C_(VPDB)=−19 per mil (Olsson,The use of Oxalic acid as a Standard. in, Radiocarbon Variations andAbsolute Chronology, Nobel Symposium, 12th Proc., John Wiley & Sons, NewYork (1970)). Mass spectrometry results, for example, measured by ASM,are calculated using the internationally agreed upon definition of 0.95times the specific activity of NBS Oxalic Acid I (SRM 4990b) normalizedto δ¹³C_(VPDB)=−19 per mil. This is equivalent to an absolute (AD 1950)¹⁴C/¹²C ratio of 1.176±0.010×10⁻¹² (Karlen et al., Arkiv Geofysik,4:465-471 (1968)). The standard calculations take into account thedifferential uptake of one isotope with respect to another, for example,the preferential uptake in biological systems of C¹² over C¹³ over C¹⁴,and these corrections are reflected as a Fm corrected for δ¹³.

An oxalic acid standard (SRM 4990b or HOx 1) was made from a crop of1955 sugar beet. Although there were 1000 lbs made, this oxalic acidstandard is no longer commercially available. The Oxalic Acid IIstandard (HOx 2; N.I.S.T designation SRM 4990 C) was made from a crop of1977 French beet molasses. In the early 1980's, a group of 12laboratories measured the ratios of the two standards. The ratio of theactivity of Oxalic acid II to 1 is 1.2933±0.001 (the weighted mean). Theisotopic ratio of HOx II is −17.8 per mille. ASTM D6866-11 suggests useof the available Oxalic Acid II standard SRM 4990 C (Hox2) for themodern standard (see discussion of original vs. currently availableoxalic acid standards in Mann, Radiocarbon, 25(2):519-527 (1983)). AFm=0% represents the entire lack of carbon-14 atoms in a material, thusindicating a fossil (for example, petroleum based) carbon source. AFm=100%, after correction for the post-1950 injection of carbon-14 intothe atmosphere from nuclear bomb testing, indicates an entirely moderncarbon source. As described herein, such a “modern” source includesbio-based sources.

As described in ASTM D6866, the percent modern carbon (pMC) can begreater than 100% because of the continuing but diminishing effects ofthe 1950s nuclear testing programs, which resulted in a considerableenrichment of carbon-14 in the atmosphere as described in ASTM D6866-11.Because all sample carbon-14 activities are referenced to a “pre-bomb”standard, and because nearly all new bio-based products are produced ina post-bomb environment, all pMC values (after correction for isotopicfraction) must be multiplied by 0.95 (as of 2010) to better reflect thetrue bio-based content of the sample. A bio-based content that isgreater than 103% suggests that either an analytical error has occurred,or that the source of bio-based carbon is more than several years old.

ASTM D6866 quantifies the bio-based content relative to the material'stotal organic content and does not consider the inorganic carbon andother non-carbon containing substances present. For example, a productthat is 50% starch-based material and 50% water would be considered tohave a Bio-derived Content=100% (50% organic content that is 100%bio-based) based on ASTM D6866. In another example, a product that is50% starch-based material, 25% petroleum-based, and 25% water would havea Bio-derived Content=66.7% (75% organic content but only 50% of theproduct is bio-based). In another example, a product that is 50% organiccarbon and is a petroleum-based product would be considered to have aBio-derived Content=0% (50% organic carbon but from fossil sources).Thus, based on the well known methods and known standards fordetermining the bio-based content of a compound or material, one skilledin the art can readily determine the bio-derived content and/or prepareddownstream products that utilize of the invention having a desiredbio-based content.

Applications of carbon-14 dating techniques to quantify bio-basedcontent of materials are known in the art (Currie et al., NuclearInstruments and Methods in Physics Research B, 172:281-287 (2000)). Forexample, carbon-14 dating has been used to quantify bio-based content interephthalate-containing materials (Colonna et al., Green Chemistry,13:2543-2548 (2011)). Notably, polypropylene terephthalate (PPT)polymers derived from renewable 1,3-propanediol and petroleum-derivedterephthalic acid resulted in Fm values near 30% (i.e., since 3/11 ofthe polymeric carbon derives from renewable 1,3-propanediol and 8/11from the fossil end member terephthalic acid) (Currie et al., supra,2000). In contrast, polybutylene terephthalate polymer derived from bothrenewable 1,4-butanediol and renewable terephthalic acid resulted inbio-based content exceeding 90% (Colonna et al., supra, 2011).

Accordingly, in some embodiments, the present invention provides2H3M4OP, p-toluate or terephthalate or a 2H3M4OP, p-toluate orterephthalate pathway intermediate that has a carbon-12, carbon-13, andcarbon-14 ratio that reflects an atmospheric carbon, also referred to asenvironmental carbon, uptake source. For example, in some aspects the2H3M4OP, p-toluate or terephthalate or a 2H3M4OP, p-toluate orterephthalate pathway intermediate can have an Fm value of at least 10%,at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, atleast 40%, at least 45%, at least 50%, at least 55%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98% or as much as 100%. In some suchembodiments, the uptake source is CO₂. In some embodiments, the presentinvention provides 2H3M4OP, p-toluate or terephthalate or a 2H3M4OP,p-toluate or terephthalate pathway intermediate that has a carbon-12,carbon-13, and carbon-14 ratio that reflects petroleum-based carbonuptake source. In this aspect, the 2H3M4OP, p-toluate or terephthalateor a 2H3M4OP, p-toluate or terephthalate pathway intermediate can havean Fm value of less than 95%, less than 90%, less than 85%, less than80%, less than 75%, less than 70%, less than 65%, less than 60%, lessthan 55%, less than 50%, less than 45%, less than 40%, less than 35%,less than 30%, less than 25%, less than 20%, less than 15%, less than10%, less than 5%, less than 2% or less than 1%. In some embodiments,the present invention provides 2H3M4OP, p-toluate or terephthalate or a2H3M4OP, p-toluate or terephthalate pathway intermediate that has acarbon-12, carbon-13, and carbon-14 ratio that is obtained by acombination of an atmospheric carbon uptake source with apetroleum-based uptake source. Using such a combination of uptakesources is one way by which the carbon-12, carbon-13, and carbon-14ratio can be varied, and the respective ratios would reflect theproportions of the uptake sources.

Further, the present invention relates to the biologically produced2H3M4OP, p-toluate or terephthalate or a 2H3M4OP, p-toluate orterephthalate pathway intermediate as disclosed herein, and to theproducts derived therefrom, wherein the 2H3M4OP, p-toluate orterephthalate or a 2H3M4OP, p-toluate or terephthalate pathwayintermediate has a carbon-12, carbon-13, and carbon-14 isotope ratio ofabout the same value as the CO₂ that occurs in the environment. Forexample, in some aspects the invention provides bio-based 2H3M4OP,p-toluate or terephthalate or a bio-based 2H3M4OP, p-toluate orterephthalate intermediate having a carbon-12 versus carbon-13 versuscarbon-14 isotope ratio of about the same value as the CO₂ that occursin the environment, or any of the other ratios disclosed herein. It isunderstood, as disclosed herein, that a product can have a carbon-12versus carbon-13 versus carbon-14 isotope ratio of about the same valueas the CO₂ that occurs in the environment, or any of the ratiosdisclosed herein, wherein the product is generated from bio-based2H3M4OP, p-toluate or terephthalate or a bio-based 2H3M4OP, p-toluate orterephthalate pathway intermediate as disclosed herein, wherein thebio-based product is chemically modified to generate a final product.Methods of chemically modifying a bio-based product of 2H3M4OP,p-toluate or terephthalate, or an intermediate thereof, to generate adesired product are well known to those skilled in the art, as describedherein. The invention further provides a chip, resin, fiber, film or anyother product described herein having a carbon-12 versus carbon-13versus carbon-14 isotope ratio of about the same value as the CO₂ thatoccurs in the environment, wherein the chip, resin, fiber, film or anyother product described herein is generated directly from or incombination with bio-based 2H3M4OP, p-toluate or terephthalate or abio-based 2H3M4OP, p-toluate or terephthalate pathway intermediate asdisclosed herein.

The terephthalate produced by the microbial organisms, pathways,fermentation process and/or isolation or purification processesdescribed herein can be used as a precursor of polymers including PET,polybutyl terephthalate (PBT), polytrimethylene terephthalate (PTT), andpolyethylene naphthalate (PEN). Accordingly, the resulting bio-derivedpolymer can contain terephthalate, comprise terephthalate, be obtainedwith terephthalate, be obtained by using terephthalate, be made by usingterephthalate, be obtained by converting terephthalate, or be obtainedby a reaction using terephthalate, wherein the terephthalate is producedby a microbial organism, pathway, fermentation process and/or isolationor purification process described herein. Moreover, in some aspects, theinvention provides use of a bio-derived polymer described herein to makea product disclosed herein. Still further, the invention provides, insome aspects, use of a product disclosed herein in making a composition,product or article.

When both a bio-based terephthalate of the invention and a secondnon-bio-based terephthalat, such as a petroleum-derived terephthalate ora therephthalate not made or obtained according to the presentinvention, are present in the same composition or product, such as apolymer, chip, fiber, resin, film or any other product described herein,the bio-based terephthalate can be present in an amount from 5% byweight to 100% by weight based on the total weight of the bio-basedterephthalate and the second non-bio-based terephthalate present in thecomposition or product. In some aspects, the bio-based terephthalate canbe from 25% by weight to 100% by weight; or alternatively, from 50% byweight to 100% by weight.

When both a polymer of the invention, i.e. a bio-derived polymer, and asecond polymer such as a petroleum based polymer or a polymer not madeor obtained by the bio-based terephthalate of the invention are presentin the same composition or product, e.g. resin, chip, fiber, film, orany other product described herein, the bio-derived polymer can bepresent in an amount from 5% by weight to 100% by weight based on thetotal weight of the bio-derived polymer and the second polymer presentin the composition or product. In some aspects, the bio-derived polymercan be present in an amount from 25% by weight to 100% by weight, oralternatively, from 50% by weight to 100% by weight.

In certain embodiments, the invention provides a PET polymer used inpolyester products and methods described herein. PET can also bereferred to as melt-phase PET resin, reactor-grade polyester orpolyester chip, e.g. PET bottle chips. A PET based polymer can be usedin the production of polyester fibers or filaments, polyester film,solid-state (bottle-grade) resins and PET engineering resins of theinvention. Polyethylene terephthalate used to make synthetic fibers isoften referred to as polyester and can be used to make bottle andpackaging, which are often referred to by its acronym PET. Products ofthe invention using polyester fibers include cloth, clothing, bedding,furniture, and carpet. Products of the invention using Bottle Grade PETinclude beverage, food and pharmaceutical containers and packaging,including beverage bottles. Polyester films or sheets include thosehaving a single layer or a multilayer, a heat-shrinkable film, hollowcontainers, formed articles, fibers, having a coating material of thesolution type, having a coating of the powder type, having a toner andhaving an adhesive. Products of the invention using polyester filminclude coated and uncoated film, and includes an Adhesion Film, aBarrier Film, a Coated Film, a Heat Sealable Film, a Lidding Film, aMatte Film, a Siliconized Release Film and/or a UV Stabilized film.Polyester film used for packaging can be coated or uncoated, and can bea Matte Film, High COF (coefficient of friction) Film, Direct ExtrudableFilm, UV Stabilized Film, Cold Seal Film, White Film, Weldable Film,thermal lamination film, and Peelable Film. Polyester film can also beused in products and uses including: a balloon; as electrical shrinktubing, and electrical wire and cable wraps; as a release liner inmultilayer ceramic capacitor (MLCC) and ceramic casting; as a layer orbacking for a photovoltaic apparatus such as a photovoltaic solar moduleor used as a part of the construction for coated flexible photovoltaicsolar module; in presentation and display media, printing and pre-pressfilm, overhead transparencies, photo-tool, medical and x-ray film andplates, printing plates, reprographic films, and digital imaging film,for example as used with a printer; for industrial applications as aRoll Leaf, Protective Face Shield or Window Box; for constructionindustry products including a shingle release liner, peel and stickrelease liner, flexible ductwork, fiberglass roofing, fiberglass tubing,carpet backing, flooring foam substrate, adhesive pads for carpet tileinstallation, acoustical panel, ceiling panel, and solar window andsafety film; and a Thermal Transfer Ribbons (TTR) for use in thermaltransfer printing. An example of such films include those under tradename Hostaphan® (Mitsubishi Polyester Film, Japan) and Mylar®, Melinex®,and Tetoron® (DuPont USA and Teijin Japan).

PET of the invention can be produced by polycondensation of ethyleneglycol with either dimethyl terephthalate or terephthalic acid of theinvention. For example, with dimethyl terephthalate, atransesterification reaction can be sued, or with terephthalic acid, anesterification reaction can be sued (Köpnick et al. “Polyesters” inUllmann's Encyclopedia of Industrial Chemistry, A21, Wiley-VCH,Weinheim, 2000). Dimethyl terephthalate can also be produced bydi-esterification of terephthalate of the invention and methanol(Sheehan, “Terephthalic Acid, Dimethyl Terephthalate, and IsophthalicAcid” in Ullmann's Encyclopedia of Industrial Chemistry, Wiley-VCH,Weinheim, 2005). Accordingly, in certain embodiments, the inventionprovides a process for obtaining polyethylene terephthalate by reactingethylene glycol with dimethyl terephthalate, wherein the dimethylterephthalate is produced from terephthalate produced by or obtainedfrom a microorganism, pathway, fermentation process and/or isolation orpurification process described herein. In another embodiment is aprocess for obtaining polyethylene terephthalate by reacting ethyleneglycol with terephthalate, wherein the terephthalate is produced by orobtained from a microorganism, pathway, fermentation process and/orisolation or purification process described herein.

Accordingly, in certain embodiments, the invention provides polyethyleneterephthalate obtained by a process using any of the microorganisms,processes and embodiments described herein. Also provided ispolyethylene terephthalate polymer obtained from terephthalate obtainedby a process with any of the microorganisms, processes and embodimentsdescribed herein claims. Still further provided is polyester chip, resinor fiber comprising or obtained by using polyethylene terephthalateobtained by a process with any of the microorganisms, processes andembodiments described herein claims. Also provided is a product, as wellas a process to obtain that product, as described herein above, e.g. apolyester cloth, carpet, film, comprising or obtained by using polyesterfiber obtained by terephthalate as disclosed herein. Also provided is aproduct, as well as a process to obtain that product, as describedherein, for example, a PET bottle or PET container or packaging,comprising or obtained by using PET chip or resin obtained byterephthalate as disclosed herein. In another embodiment are PET bottlechips, or method of their making, comprising or obtained by using PETobtained by a process as described herein. Further disclosed is a PETbottle, container or package comprising or obtained by using PET bottlechips obtained as described herein.

In certain embodiments, the invention provides a polybutyl terephthalate(PBT) polymer that can be used in PBT products and processes of theinvention as described herein. PBT polymer can be used in moldedarticles, for example, injection-molded products and parts, includingautomotive parts, in extrusion resin, in electrical and automotiveparts, and in casings, for example, power tool casings. Products includethose containing PBT sold under the trade name Advanite (SASA), Anjacom(Almaak International), Arnite (DSM), Celanex, Vandar polyester alloy(Ticona), Duranex (Polyplastics), Crastin (DuPont), Pocan (Lanxess),Ultradur (BASF), Valox (SABIC Innovative Plastics), Schuladur (A.Schulman), Later (LATI), Kebater (BARLOG plastics), VESTODUR (EvonikDegussa), and ENVIRON® (Enviroplas). A PBT polymer of the invention canbe produced by reaction of 1,4-butanediol with terephthalic acid of theinvention, for example by esterification. Accordingly, in oneembodiment, the invention provides a process for obtaining PBT byreacting 1,4-butanediol with terephthalate where the terephthalate isobtained by or produced from a microrganism, pathway, fermentationprocess and/or isolation or purification process described herein.

In certain embodiments, the invention provides polytrimethyleneterephthalate (PTT) polymer that can be used in PTT products andprocesses of the invention as described herein. A PTT polymer can beused in fibers to make products, such as cloth, clothing and carpet, orbulk products such as, for example, chips for the manufacture ofbottles. PTT can be prepared by the esterification of 1,3-propanediolwith terephthalic acid, or by transesterification of dimethylterephthalate, where the terephthalate is obtained by or produced from amicroorganism, pathway, fermentation process and/or isolation orpurification process described herein. Products and processes of theinvention include those that comprise PBT polymer that is sold under thetrade name Sorona (DuPont), and includes co-polymers, for example, apoly(trimethylene terephthalate) copolymer (e.g. Tritan by Eastman) andas used to produce Nalgene bottles.

The polymers of the present invention may further comprise additionalcompounds. Additional compounds include a solvent; one or more otherbio-derived polymers described herein; one or more resins includingpolyester, aliphatic polyester resins, thermoplastic polyesterelastomers, polyolefins, polystyrenes, acrylonitrile-butadiene-styrenecopolymers, polymethyl methacrylate, polysulfones, polyethers, phenoxyresins, polyphenylene oxides, thermoplastic resins such as polyethylene,polypropylene, acrylic resins, polycarbonates, polyamides, polyphenylenesulfide, polyethylene terephthalate, liquid crystalline polyesters,polyacetals and polyphenylene oxide and thermosetting resins such asphenol resins, melamine resins, silicone resins and epoxy resins (whichmay be added when a molded article is prepared); and/or one or moreauxiliary agents. Auxiliary agents can be added during thepolymerization to form the bio-derived polymer or after thepolymerization. Auxiliary agents include those for molding and additivessuch as fillers, coloring agents, reinforcing materials,surface-smoothing agents, leveling agents, accelerators for a curingreaction, photostabilizers, ultraviolet absorbers, plasticizers,antioxidants, extenders, delustering agents, agents for adjustingdrying, antistatic agents, agents for preventing precipitating,surfactants, agents for improving flow, drying oils, waxes andthermoplastic oligomers, stabilizers including heat and UV stabilizers,lubricants, catalyst deactivators, nucleating agents for crystallizationand promoters for crystallization. To provide the bio-polymer withdesired properties, ultraviolet absorbents, stabilizers such asweathering stabilizers, coloring agents such as dyes, antistatic agents,foaming agents, plasticizers and impact resistance improvers can beadded. Reinforcing fillers added to the resin of the present inventionare not particularly limited, examples include inorganic fibers such asglass fiber, carbon fiber, silica-alumina fiber, zirconia fiber, boronfiber, boron nitride fiber, silicon nitride potassium titanate fiber andmetal fibers; and organic fibers such as aromatic polyamide fibers andfluoro-resin fibers. Other fillers include inorganic fillers having aplate shape, ceramic beads, wollastonite, talc, clay, mica, zeolite,kaolin, potassium titanate, barium sulfate, titanium oxide, siliconoxide, aluminum oxide and magnesium hydroxide. By adding an inorganicfiller having a plate shape, anisotropy and warp in the molded articlecan be decreased. Preferable examples of the inorganic filler having aplate shape include glass flakes, mica and metal foils. Among theseinorganic fillers having a plate shape, glass flakes are more preferablyused. Antioxidants include phenolic antioxidants, sterically hinderedphenols and/or phosphites, hydroquinones, aromatic secondary amines,such as diphenylamines, and various substituted representatives of thesegroups. UV stabilizers include various substituted resorcinols,salicylates, benzotriazoles, and benzophenones. Coloring agents that canbe added comprise inorganic and organic pigments, and also dyes, such asnigrosin and anthraquinones. Lubricants and mold-release agents includelong-chain fatty acids (e.g. stearic acid or behenic acid), salts ofthese (e.g. Ca stearate or Zn stearate), or montan waxes (mixtures madeof straight-chain, saturated carboxylic acids having chain lengths offrom 28 to 32 carbon atoms), Ca montanate or Na montanate,low-molecular-weight polyethylene waxes or low-molecular-weightpolypropylene waxes.

The bio-derived polymers of the present invention can be molded inaccordance with a conventional process suitable for each application.For example, molded articles can be prepared in accordance with theconventional injection molding process and sheets and films can beprepared in accordance with the extrusion process or the castingprocess. Formed articles can be prepared in accordance with theextrusion-expansion process or the expansion-in-mold process. A moldingprocess conventionally used for thermoplastic resins such as theinjection molding, the blow molding, the extrusion molding and thecompression molding can be applied.

In some embodiments, the invention provides chip, resin, fiber, film orany other product described herein comprising bio-based 2H3M4OP,p-toluate or terephthalate, or a bio-based 2H3M4OP, p-toluate orterephthalate pathway intermediate, wherein the bio-based 2H3M4OP,p-toluate or terephthalate, or bio-based 2H3M4OP, p-toluate orterephthalate pathway intermediate includes all or part of the 2H3M4OP,p-toluate or terephthalate, or 2H3M4OP, p-toluate or terephthalatepathway intermediate used in the production of chip, resin, fiber, filmor any other product described herein. For example, the final chip,resin, fiber, film or any other product described herein can contain thebio-based 2H3M4OP, p-toluate or terephthalate, 2H3M4OP, p-toluate orterephthalate pathway intermediate, or a portion thereof that is theresult of the manufacturing of chip, resin, fiber, film or any otherproduct described herein. Such manufacturing can include chemicallyreacting the bio-based 2H3M4OP, p-toluate or terephthalate or bio-based2H3M4OP, p-toluate or terephthalate pathway intermediate (e.g. chemicalconversion, chemical functionalization, chemical coupling, oxidation,reduction, polymerization, copolymerization and the like) into the finalchip, resin, fiber, film or any other product described herein. Thus, insome aspects, the invention provides a bio-derived chip, resin, fiber,film or any other product described herein comprising at least 2%, atleast 3%, at least 5%, at least 10%, at least 15%, at least 20%, atleast 25%, at least 30%, at least 35%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 80%, at least 90%, at least 95%, atleast 98% or 100% bio-based 2H3M4OP, p-toluate or terephthalate orbio-based 2H3M4OP, p-toluate or terephthalate pathway intermediate asdisclosed herein.

Additionally, in some embodiments, the invention provides a compositionhaving a bio-based 2H3M4OP, p-toluate or terephthalate or 2H3M4OP,p-toluate or terephthalate pathway intermediate disclosed herein and acompound other than the bio-based 2H3M4OP, p-toluate or terephthalate or2H3M4OP, p-toluate or terephthalate pathway intermediate. For example,in some aspects, the invention provides a bio-derived chip, resin,fiber, film or any other product described herein wherein the 2H3M4OP,p-toluate or terephthalate, or 2H3M4OP, p-toluate or terephthalatepathway intermediate used in its production is a combination ofbio-based and petroleum derived 2H3M4OP, p-toluate or terephthalate, or2H3M4OP, p-toluate or terephthalate pathway intermediate. For example, abio-derived chip, resin, fiber, film or any other product describedherein can be produced using 50% bio-based 2H3M4OP, p-toluate orterephthalate and 50% petroleum derived 2H3M4OP, p-toluate orterephthalate or other desired ratios such as 60%/40%, 70%/30%, 80%/20%,90%/10%, 95%/5%, 100%/0%, 40%/60%, 30%/70%, 20%/80%, 10%/90% ofbio-based/petroleum derived precursors, so long as at least a portion ofthe product comprises a bio-based product produced by the microbialorganisms disclosed herein. It is understood that methods for producingchip, resin, fiber, film or any other product described herein using thebio-based 2H3M4OP, p-toluate or terephthalate or bio-based 2H3M4OP,p-toluate or terephthalate pathway intermediate of the invention arewell known in the art.

The culture conditions can include, for example, liquid cultureprocedures as well as fermentation and other large scale cultureprocedures. As described herein, particularly useful yields of thebiosynthetic products of the invention can be obtained under anaerobicor substantially anaerobic culture conditions.

As described herein, one exemplary growth condition for achievingbiosynthesis of 2H3M4OP, p-toluate or terephthalate includes anaerobicculture or fermentation conditions. In certain embodiments, thenon-naturally occurring microbial organisms of the invention can besustained, cultured or fermented under anaerobic or substantiallyanaerobic conditions. Briefly, an anaerobic condition refers to anenvironment devoid of oxygen. Substantially anaerobic conditionsinclude, for example, a culture, batch fermentation or continuousfermentation such that the dissolved oxygen concentration in the mediumremains between 0 and 10% of saturation. Substantially anaerobicconditions also includes growing or resting cells in liquid medium or onsolid agar inside a sealed chamber maintained with an atmosphere of lessthan 1% oxygen. The percent of oxygen can be maintained by, for example,sparging the culture with an N₂/CO₂ mixture or other suitable non-oxygengas or gases.

The culture conditions described herein can be scaled up and growncontinuously for manufacturing of 2H3M4OP, p-toluate or terephthalate.Exemplary growth procedures include, for example, fed-batch fermentationand batch separation; fed-batch fermentation and continuous separation,or continuous fermentation and continuous separation. All of theseprocesses are well known in the art. Fermentation procedures areparticularly useful for the biosynthetic production of commercialquantities of 2H3M4OP, p-toluate or terephthalate. Generally, and aswith non-continuous culture procedures, the continuous and/ornear-continuous production of 2H3M4OP, p-toluate or terephthalate willinclude culturing a non-naturally occurring 2H3M4OP, p-toluate orterephthalate producing organism of the invention in sufficientnutrients and medium to sustain and/or nearly sustain growth in anexponential phase. Continuous culture under such conditions can include,for example, growth for 1 day, 2, 3, 4, 5, 6 or 7 days or more.Additionally, continuous culture can include longer time periods of 1week, 2, 3, 4 or 5 or more weeks and up to several months.Alternatively, organisms of the invention can be cultured for hours, ifsuitable for a particular application. It is to be understood that thecontinuous and/or near-continuous culture conditions also can includeall time intervals in between these exemplary periods. It is furtherunderstood that the time of culturing the microbial organism of theinvention is for a sufficient period of time to produce a sufficientamount of product for a desired purpose.

Fermentation procedures are well known in the art. Briefly, fermentationfor the biosynthetic production of 2H3M4OP, p-toluate or terephthalatecan be utilized in, for example, fed-batch fermentation and batchseparation; fed-batch fermentation and continuous separation, orcontinuous fermentation and continuous separation. Examples of batch andcontinuous fermentation procedures are well known in the art.

In addition to the above fermentation procedures using the 2H3M4OP,p-toluate or terephthalate producers of the invention for continuousproduction of substantial quantities of 2H3M4OP, p-toluate orterephthalate, the 2H3M4OP, p-toluate or terephthalate producers alsocan be, for example, simultaneously subjected to chemical synthesisand/or enzymatic procedures to convert the product to other compounds orthe product can be separated from the fermentation culture andsequentially subjected to chemical and/or enzymatic conversion toconvert the product to other compounds, if desired.

To generate better producers, metabolic modeling can be utilized tooptimize growth conditions. Modeling can also be used to design geneknockouts that additionally optimize utilization of the pathway (see,for example, U.S. patent publications US 2002/0012939, US 2003/0224363,US 2004/0029149, US 2004/0072723, US 2003/0059792, US 2002/0168654 andUS 2004/0009466, and U.S. Pat. No. 7,127,379). Modeling analysis allowsreliable predictions of the effects on cell growth of shifting themetabolism towards more efficient production of 2H3M4OP, p-toluate orterephthalate.

One computational method for identifying and designing metabolicalterations favoring biosynthesis of a desired product is the OptKnockcomputational framework (Burgard et al., Biotechnol. Bioeng. 84:647-657(2003)). OptKnock is a metabolic modeling and simulation program thatsuggests gene deletion or disruption strategies that result ingenetically stable microorganisms which overproduce the target product.Specifically, the framework examines the complete metabolic and/orbiochemical network of a microorganism in order to suggest geneticmanipulations that force the desired biochemical to become an obligatorybyproduct of cell growth. By coupling biochemical production with cellgrowth through strategically placed gene deletions or other functionalgene disruption, the growth selection pressures imposed on theengineered strains after long periods of time in a bioreactor lead toimprovements in performance as a result of the compulsory growth-coupledbiochemical production. Lastly, when gene deletions are constructedthere is a negligible possibility of the designed strains reverting totheir wild-type states because the genes selected by OptKnock are to becompletely removed from the genome. Therefore, this computationalmethodology can be used to either identify alternative pathways thatlead to biosynthesis of a desired product or used in connection with thenon-naturally occurring microbial organisms for further optimization ofbiosynthesis of a desired product.

Briefly, OptKnock is a term used herein to refer to a computationalmethod and system for modeling cellular metabolism. The OptKnock programrelates to a framework of models and methods that incorporate particularconstraints into flux balance analysis (FBA) models. These constraintsinclude, for example, qualitative kinetic information, qualitativeregulatory information, and/or DNA microarray experimental data.OptKnock also computes solutions to various metabolic problems by, forexample, tightening the flux boundaries derived through flux balancemodels and subsequently probing the performance limits of metabolicnetworks in the presence of gene additions or deletions. OptKnockcomputational framework allows the construction of model formulationsthat allow an effective query of the performance limits of metabolicnetworks and provides methods for solving the resulting mixed-integerlinear programming problems. The metabolic modeling and simulationmethods referred to herein as OptKnock are described in, for example,U.S. publication 2002/0168654, filed Jan. 10, 2002, in InternationalPatent No. PCT/US02/00660, filed Jan. 10, 2002, and U.S. publication2009/0047719, filed Aug. 10, 2007.

Another computational method for identifying and designing metabolicalterations favoring biosynthetic production of a product is a metabolicmodeling and simulation system termed SimPheny®. This computationalmethod and system is described in, for example, U.S. publication2003/0233218, filed Jun. 14, 2002, and in International PatentApplication No. PCT/US03/18838, filed Jun. 13, 2003. SimPheny® is acomputational system that can be used to produce a network model insilico and to simulate the flux of mass, energy or charge through thechemical reactions of a biological system to define a solution spacethat contains any and all possible functionalities of the chemicalreactions in the system, thereby determining a range of allowedactivities for the biological system. This approach is referred to asconstraints-based modeling because the solution space is defined byconstraints such as the known stoichiometry of the included reactions aswell as reaction thermodynamic and capacity constraints associated withmaximum fluxes through reactions. The space defined by these constraintscan be interrogated to determine the phenotypic capabilities andbehavior of the biological system or of its biochemical components.

These computational approaches are consistent with biological realitiesbecause biological systems are flexible and can reach the same result inmany different ways. Biological systems are designed throughevolutionary mechanisms that have been restricted by fundamentalconstraints that all living systems must face. Therefore,constraints-based modeling strategy embraces these general realities.Further, the ability to continuously impose further restrictions on anetwork model via the tightening of constraints results in a reductionin the size of the solution space, thereby enhancing the precision withwhich physiological performance or phenotype can be predicted.

Given the teachings and guidance provided herein, those skilled in theart will be able to apply various computational frameworks for metabolicmodeling and simulation to design and implement biosynthesis of adesired compound in host microbial organisms. Such metabolic modelingand simulation methods include, for example, the computational systemsexemplified above as SimPheny® and OptKnock. For illustration of theinvention, some methods are described herein with reference to theOptKnock computation framework for modeling and simulation. Thoseskilled in the art will know how to apply the identification, design andimplementation of the metabolic alterations using OptKnock to any ofsuch other metabolic modeling and simulation computational frameworksand methods well known in the art.

The methods described above will provide one set of metabolic reactionsto disrupt. Elimination of each reaction within the set or metabolicmodification can result in a desired product as an obligatory productduring the growth phase of the organism. Because the reactions areknown, a solution to the bilevel OptKnock problem also will provide theassociated gene or genes encoding one or more enzymes that catalyze eachreaction within the set of reactions. Identification of a set ofreactions and their corresponding genes encoding the enzymesparticipating in each reaction is generally an automated process,accomplished through correlation of the reactions with a reactiondatabase having a relationship between enzymes and encoding genes.

Once identified, the set of reactions that are to be disrupted in orderto achieve production of a desired product are implemented in the targetcell or organism by functional disruption of at least one gene encodingeach metabolic reaction within the set. One particularly useful means toachieve functional disruption of the reaction set is by deletion of eachencoding gene. However, in some instances, it can be beneficial todisrupt the reaction by other genetic aberrations including, forexample, mutation, deletion of regulatory regions such as promoters orcis binding sites for regulatory factors, or by truncation of the codingsequence at any of a number of locations. These latter aberrations,resulting in less than total deletion of the gene set can be useful, forexample, when rapid assessments of the coupling of a product are desiredor when genetic reversion is less likely to occur.

To identify additional productive solutions to the above describedbilevel OptKnock problem which lead to further sets of reactions todisrupt or metabolic modifications that can result in the biosynthesis,including growth-coupled biosynthesis of a desired product, anoptimization method, termed integer cuts, can be implemented. Thismethod proceeds by iteratively solving the OptKnock problem exemplifiedabove with the incorporation of an additional constraint referred to asan integer cut at each iteration. Integer cut constraints effectivelyprevent the solution procedure from choosing the exact same set ofreactions identified in any previous iteration that obligatorily couplesproduct biosynthesis to growth. For example, if a previously identifiedgrowth-coupled metabolic modification specifies reactions 1, 2, and 3for disruption, then the following constraint prevents the samereactions from being simultaneously considered in subsequent solutions.The integer cut method is well known in the art and can be founddescribed in, for example, Burgard et al., Biotechnol. Prog. 17:791-797(2001). As with all methods described herein with reference to their usein combination with the OptKnock computational framework for metabolicmodeling and simulation, the integer cut method of reducing redundancyin iterative computational analysis also can be applied with othercomputational frameworks well known in the art including, for example,SimPheny®.

The methods exemplified herein allow the construction of cells andorganisms that biosynthetically produce a desired product, including theobligatory coupling of production of a target biochemical product togrowth of the cell or organism engineered to harbor the identifiedgenetic alterations. Therefore, the computational methods describedherein allow the identification and implementation of metabolicmodifications that are identified by an in silico method selected fromOptKnock or SimPheny®. The set of metabolic modifications can include,for example, addition of one or more biosynthetic pathway enzymes and/orfunctional disruption of one or more metabolic reactions including, forexample, disruption by gene deletion.

As discussed above, the OptKnock methodology was developed on thepremise that mutant microbial networks can be evolved towards theircomputationally predicted maximum-growth phenotypes when subjected tolong periods of growth selection. In other words, the approach leveragesan organism's ability to self-optimize under selective pressures. TheOptKnock framework allows for the exhaustive enumeration of genedeletion combinations that force a coupling between biochemicalproduction and cell growth based on network stoichiometry. Theidentification of optimal gene/reaction knockouts requires the solutionof a bilevel optimization problem that chooses the set of activereactions such that an optimal growth solution for the resulting networkoverproduces the biochemical of interest (Burgard et al., Biotechnol.Bioeng. 84:647-657 (2003)).

An in silico stoichiometric model of E. coli metabolism can be employedto identify essential genes for metabolic pathways as exemplifiedpreviously and described in, for example, U.S. patent publications US2002/0012939, US 2003/0224363, US 2004/0029149, US 2004/0072723, US2003/0059792, US 2002/0168654 and US 2004/0009466, and in U.S. Pat. No.7,127,379. As disclosed herein, the OptKnock mathematical framework canbe applied to pinpoint gene deletions leading to the growth-coupledproduction of a desired product. Further, the solution of the bilevelOptKnock problem provides only one set of deletions. To enumerate allmeaningful solutions, that is, all sets of knockouts leading togrowth-coupled production formation, an optimization technique, termedinteger cuts, can be implemented. This entails iteratively solving theOptKnock problem with the incorporation of an additional constraintreferred to as an integer cut at each iteration, as discussed above.

As disclosed herein, a nucleic acid encoding a desired activity of a2H3M4OP, p-toluate or terephthalate pathway can be introduced into ahost organism. In some cases, it can be desirable to modify an activityof a 2H3M4OP, p-toluate or terephthalate pathway enzyme or protein toincrease production of 2H3M4OP, p-toluate or terephthalate. For example,known mutations that increase the activity of a protein or enzyme can beintroduced into an encoding nucleic acid molecule. Additionally,optimization methods can be applied to increase the activity of anenzyme or protein and/or decrease an inhibitory activity, for example,decrease the activity of a negative regulator.

One such optimization method is directed evolution. Directed evolutionis a powerful approach that involves the introduction of mutationstargeted to a specific gene in order to improve and/or alter theproperties of an enzyme. Improved and/or altered enzymes can beidentified through the development and implementation of sensitivehigh-throughput screening assays that allow the automated screening ofmany enzyme variants (for example, >10⁴). Iterative rounds ofmutagenesis and screening typically are performed to afford an enzymewith optimized properties. Computational algorithms that can help toidentify areas of the gene for mutagenesis also have been developed andcan significantly reduce the number of enzyme variants that need to begenerated and screened. Numerous directed evolution technologies havebeen developed (for reviews, see Hibbert et al., Biomol. Eng 22:11-19(2005); Huisman and Lalonde, In Biocatalysis in the pharmaceutical andbiotechnology industries pgs. 717-742 (2007), Patel (ed.), CRC Press;Otten and Quax. Biomol. Eng 22:1-9 (2005); and Sen et al., Appl Biochem.Biotechnol 143:212-223 (2007)) to be effective at creating diversevariant libraries, and these methods have been successfully applied tothe improvement of a wide range of properties across many enzymeclasses. Enzyme characteristics that have been improved and/or alteredby directed evolution technologies include, for example:selectivity/specificity, for conversion of non-natural substrates;temperature stability, for robust high temperature processing; pHstability, for bioprocessing under lower or higher pH conditions;substrate or product tolerance, so that high product titers can beachieved; binding (K_(m)), including broadening substrate binding toinclude non-natural substrates; inhibition (K_(i)), to remove inhibitionby products, substrates, or key intermediates; activity (kcat), toincreases enzymatic reaction rates to achieve desired flux; expressionlevels, to increase protein yields and overall pathway flux; oxygenstability, for operation of air sensitive enzymes under aerobicconditions; and anaerobic activity, for operation of an aerobic enzymein the absence of oxygen.

A number of exemplary methods have been developed for the mutagenesisand diversification of genes to target desired properties of specificenzymes. Such methods are well known to those skilled in the art. Any ofthese can be used to alter and/or optimize the activity of a 2H3M4OP,p-toluate or terephthalate pathway enzyme or protein. Such methodsinclude, but are not limited to EpPCR, which introduces random pointmutations by reducing the fidelity of DNA polymerase in PCR reactions(Pritchard et al., J Theor. Biol. 234:497-509 (2005)); Error-proneRolling Circle Amplification (epRCA), which is similar to epPCR except awhole circular plasmid is used as the template and random 6-mers withexonuclease resistant thiophosphate linkages on the last 2 nucleotidesare used to amplify the plasmid followed by transformation into cells inwhich the plasmid is re-circularized at tandem repeats (Fujii et al.,Nucleic Acids Res. 32:e145 (2004); and Fujii et al., Nat. Protoc.1:2493-2497 (2006)); DNA or Family Shuffling, which typically involvesdigestion of two or more variant genes with nucleases such as Dnase I orEndoV to generate a pool of random fragments that are reassembled bycycles of annealing and extension in the presence of DNA polymerase tocreate a library of chimeric genes (Stemmer, Proc Natl Acad Sci USA91:10747-10751 (1994); and Stemmer, Nature 370:389-391 (1994));Staggered Extension (StEP), which entails template priming followed byrepeated cycles of 2 step PCR with denaturation and very short durationof annealing/extension (as short as 5 sec) (Zhao et al., Nat.Biotechnol. 16:258-261 (1998)); Random Priming Recombination (RPR), inwhich random sequence primers are used to generate many short DNAfragments complementary to different segments of the template (Shao etal., Nucleic Acids Res 26:681-683 (1998)).

Additional methods include Heteroduplex Recombination, in whichlinearized plasmid DNA is used to form heteroduplexes that are repairedby mismatch repair (Volkov et al, Nucleic Acids Res. 27:e18 (1999); andVolkov et al., Methods Enzymol. 328:456-463 (2000)); RandomChimeragenesis on Transient Templates (RACHITT), which employs Dnase Ifragmentation and size fractionation of single stranded DNA (ssDNA)(Coco et al., Nat. Biotechnol. 19:354-359 (2001)); Recombined Extensionon Truncated templates (RETT), which entails template switching ofunidirectionally growing strands from primers in the presence ofunidirectional ssDNA fragments used as a pool of templates (Lee et al.,J. Molec. Catalysis 26:119-129 (2003)); Degenerate Oligonucleotide GeneShuffling (DOGS), in which degenerate primers are used to controlrecombination between molecules; (Bergquist and Gibbs, Methods Mol. Biol352:191-204 (2007); Bergquist et al., Biomol. Eng 22:63-72 (2005); Gibbset al., Gene 271:13-20 (2001)); Incremental Truncation for the Creationof Hybrid Enzymes (ITCHY), which creates a combinatorial library with 1base pair deletions of a gene or gene fragment of interest (Ostermeieret al., Proc. Natl. Acad. Sci. USA 96:3562-3567 (1999); and Ostermeieret al., Nat. Biotechnol. 17:1205-1209 (1999)); Thio-IncrementalTruncation for the Creation of Hybrid Enzymes (THIO-ITCHY), which issimilar to ITCHY except that phosphothioate dNTPs are used to generatetruncations (Lutz et al., Nucleic Acids Res 29:E16 (2001)); SCRATCHY,which combines two methods for recombining genes, ITCHY and DNAshuffling (Lutz et al., Proc. Natl. Acad. Sci. USA 98:11248-11253(2001)); Random Drift Mutagenesis (RNDM), in which mutations made viaepPCR are followed by screening/selection for those retaining usableactivity (Bergquist et al., Biomol. Eng. 22:63-72 (2005)); SequenceSaturation Mutagenesis (SeSaM), a random mutagenesis method thatgenerates a pool of random length fragments using random incorporationof a phosphothioate nucleotide and cleavage, which is used as a templateto extend in the presence of “universal” bases such as inosine, andreplication of an inosine-containing complement gives random baseincorporation and, consequently, mutagenesis (Wong et al., Biotechnol.J. 3:74-82 (2008); Wong et al., Nucleic Acids Res. 32:e26 (2004); andWong et al., Anal. Biochem. 341:187-189 (2005)); Synthetic Shuffling,which uses overlapping oligonucleotides designed to encode “all geneticdiversity in targets” and allows a very high diversity for the shuffledprogeny (Ness et al., Nat. Biotechnol. 20:1251-1255 (2002)); NucleotideExchange and Excision Technology NexT, which exploits a combination ofdUTP incorporation followed by treatment with uracil DNA glycosylase andthen piperidine to perform endpoint DNA fragmentation (Muller et al.,Nucleic Acids Res. 33:e117 (2005)).

Further methods include Sequence Homology-Independent ProteinRecombination (SHIPREC), in which a linker is used to facilitate fusionbetween two distantly related or unrelated genes, and a range ofchimeras is generated between the two genes, resulting in libraries ofsingle-crossover hybrids (Sieber et al., Nat. Biotechnol. 19:456-460(2001)); Gene Site Saturation Mutagenesis™ (GSSM™), in which thestarting materials include a supercoiled double stranded DNA (dsDNA)plasmid containing an insert and two primers which are degenerate at thedesired site of mutations (Kretz et al., Methods Enzymol. 388:3-11(2004)); Combinatorial Cassette Mutagenesis (CCM), which involves theuse of short oligonucleotide cassettes to replace limited regions with alarge number of possible amino acid sequence alterations (Reidhaar-Olsonet al. Methods Enzymol. 208:564-586 (1991); and Reidhaar-Olson et al.Science 241:53-57 (1988)); Combinatorial Multiple Cassette Mutagenesis(CMCM), which is essentially similar to CCM and uses epPCR at highmutation rate to identify hot spots and hot regions and then extensionby CMCM to cover a defined region of protein sequence space (Reetz etal., Angew. Chem. Int. Ed Engl. 40:3589-3591 (2001)); the MutatorStrains technique, in which conditional is mutator plasmids, utilizingthe mutD5 gene, which encodes a mutant subunit of DNA polymerase III, toallow increases of 20 to 4000-X in random and natural mutation frequencyduring selection and block accumulation of deleterious mutations whenselection is not required (Selifonova et al., Appl. Environ. Microbiol.67:3645-3649 (2001)); Low et al., J. Mol. Biol. 260:359-3680 (1996)).

Additional exemplary methods include Look-Through Mutagenesis (LTM),which is a multidimensional mutagenesis method that assesses andoptimizes combinatorial mutations of selected amino acids (Rajpal etal., Proc. Natl. Acad. Sci. USA 102:8466-8471 (2005)); Gene Reassembly,which is a DNA shuffling method that can be applied to multiple genes atone time or to create a large library of chimeras (multiple mutations)of a single gene (Tunable GeneReassembly™ (TGR™) Technology supplied byVerenium Corporation), in Silico Protein Design Automation (PDA), whichis an optimization algorithm that anchors the structurally definedprotein backbone possessing a particular fold, and searches sequencespace for amino acid substitutions that can stabilize the fold andoverall protein energetics, and generally works most effectively onproteins with known three-dimensional structures (Hayes et al., Proc.Natl. Acad. Sci. USA 99:15926-15931 (2002)); and Iterative SaturationMutagenesis (ISM), which involves using knowledge of structure/functionto choose a likely site for enzyme improvement, performing saturationmutagenesis at chosen site using a mutagenesis method such as StratageneQuikChange (Stratagene; San Diego Calif.), screening/selecting fordesired properties, and, using improved clone(s), starting over atanother site and continue repeating until a desired activity is achieved(Reetz et al., Nat. Protoc. 2:891-903 (2007); and Reetz et al., Angew.Chem. Int. Ed Engl. 45:7745-7751 (2006)).

Any of the aforementioned methods for mutagenesis can be used alone orin any combination. Additionally, any one or combination of the directedevolution methods can be used in conjunction with adaptive evolutiontechniques, as described herein.

Isolation (Purification) Process of the Invention

Provided herein are processes for isolating a bio-based aromaticcarboxylic acid such as, for example, p-toluic acid or terephthalicacid, from a culture medium, wherein the processes comprise lowering thepH of the culture medium to produce an aromatic carboxylic acidprecipitate. In certain embodiments, lowering the pH of the culturemedium comprises contacting the culture medium with carbon dioxide tolower the pH of the culture medium. In certain embodiments, a process asprovided herein further comprises culturing a non-naturally occurringmicrobial organism in a culture medium at a pH sufficient to produce abio-based aromatic carboxylic acid which will be in an anionic, solubleform in the culture medium.

As used herein, a “bio-based aromatic carboxylic acid” means an aromaticcarboxylic acid made biosynthetically from a non-naturally occurringmicrobial organism.

An aromatic carboxylic acid such as terephthalic acid can beprecipitated directly out of a culture medium to substantially depletethe medium of the acid or its conjugate anion. In certain embodiments,the bio-based aromatic carboxylic acid anion can have an ammoniumcounterion in the culture medium. Moreover, such embodiments wherecarbon dioxide is used to lower the pH to produce a bio-based aromaticacid precipitate, it will be understood that ammonium carbonate willresult which can be easily recovered as ammonia and carbon dioxide,which components may be separated, recovered and recycled. In certainembodiments, the carbon dioxide source for acidifying the culture mediumwill be that collected as produced from culturing the non-naturallyoccurring microbial organism.

Culturing the Microbial Organism to Produce the Aromatic CarboxylateAnion

In certain embodiments, the aromatic carboxylate anion is produced by anindirect semi-synthetic route, whereby muconate is biosynthesized in anon-naturally occurring microbial organism from simple carbohydratefeedstocks, which in turn provides a viable synthetic route to thearomatic carboxylate anion, for example, terephthalate, as disclosed inU.S. Patent Publication No. US 2011/0124911 A1, hereby incorporated byreference in its entirety for all purposes. In particular, thesepathways provide trans,trans-muconate or cis,trans-muconatebiocatalytically from simple sugars. The all trans or cis,trans isomerof muconate may then be converted to terephthalate in a two step processvia inverse electron demand Diels-Alder reaction with acetylene followedby oxidation in air or oxygen. The Diels-Alder reaction between muconateand acetylene proceeds to form cyclohexa-2,5-diene-1,4-dicarboxylate(P1) (see FIG. 1 of U.S. Patent Publication No. US 2011/0124911 A1,hereby incorporated by reference). Subsequent exposure to air or oxygenrapidly converts P1 to terephthalate.

In certain embodiments, the Diels-Alder reaction between muconate andacetylene can be performed in the culture medium. Optionally, theculture medium can be filtered, for example, to remove cells of theorganism, prior to adding acetylene.

In certain embodiments, the pH sufficient to maintain the muconate insoluble form is between about 5.0-9.0, between about 5.5-9.0, betweenabout 6.0-9.0, between about 6.5-9.0, between about 7.0-9.0, betweenabout 5.5-8.0, between about 6.0-8.0, or between about 6.5-8.0 pH units.In certain embodiments, the pH sufficient to maintain the muconate insoluble form is about 5.5, about 6.0, about 6.5, about 7.0, about 7.5,about 8.0, or about 8.5 pH units. In certain embodiments, the pHsufficient to maintain the muconate in soluble form is about 7.0 pHunits. In certain embodiments, the pH sufficient to maintain themuconate in soluble form is a neutral pH, for example a pH of about 7.0typically bounded on its lower end by pH of about 5.0, 5.5 or 6.0 and onits upper end by a pH of about 9.0, 8.5 or 8.0.

The pH of the culture medium can be maintained at a desired pH, forexample, a pH between about 5.0-9.0, by addition of a base, such as NaOHor other bases, or acid, as needed to maintain the culture medium at thedesired pH. It will be understood that a pH between about 5.0-9.0 can,for example, be conducive for optimal culturing conditions to produce adesired product (e.g., a bio-based aromatic carboxylate anion or aprecursor) and/or to produce an anionic form in a soluble form inculture medium. In certain embodiments, a base is added to the culturemedium in sufficient quantities to maintain muconate in soluble form. Incertain embodiments, a base is added to the culture medium in sufficientquantities to maintain the culture medium at a pH between about 5.0-9.0.

In certain embodiments, following the Diels-Alder reaction, the culturemedium can be separated from the cells and/or any non-soluble materialby, for example, centrifugation or membrane filtration, to provide acell-free medium or broth comprising the terephthalate, prior to thecontacting step (described herein and in section “Acidification of theCulture Medium to Precipitate the Aromatic Carboxylic Acid”). Forconvenience, the culture medium or broth separated from the non-solublematerials will be termed “cell-free.” Those skilled in the art willunderstand that medium and broth can be used interchangeably throughoutto refer to a liquid or gel designed to support the growth of thenon-naturally occurring microbial organism.

In certain embodiments, the aromatic carboxylate anion is produced by adirect biosynthetic route, whereby the aromatic carboxylate anion, forexample, p-toluate or terephthalate, is biosynthesized in anon-naturally occurring microbial organism from simple carbohydratefeedstocks, as disclosed in U.S. Patent Publication No. US 2011/0207185A1, which is hereby incorporated by reference in its entirety for allpurposes.

Exemplary biosynthetic pathways include, for example, the conversion oferythrose-4-phosphate to (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate(2H3M4OP), also known as 3-hydroxy-2-methyl butanal-4-phosphate, in sixenzymatic steps (see FIG. 1). In one alternative,4,5-dihydroxy-2-oxopentanoate is converted to 2H3M4OP using one or bothof the pathways described in FIG. 2. In another alternative,glyceraldehyde-3-phosphate (G3P) and pyruvate are converted to 2H3M4OPin three enzymatic steps (see FIG. 3). The 2H3M4OP intermediate can besubsequently transformed to p-toluate by enzymes in the shikimatepathway (see FIG. 4). p-Toluate can be further converted toterephthalate by a microorganism (see FIG. 5). The purification methodof the invention (exemplified in FIG. 6) can also be applied tobiosynthetic pathways for aromatic carboxylic acid and terephthalic acidproduction described in WIPO patent publications WO/2009/120457A2entitled “Bio-Based Polyethylene Terephthalate Polymer And Method OfMaking The Same”, WO/2011/094131A1 entitled “Microorganisms And MethodsFor The Biosynthesis Of P-Toluate And Terephthalate”, andWO/2011/017560A1 entitled “Semi-Synthetic Terephthalic Acid ViaMicroorganisms That Produce Muconic Acid” and U.S. Pat. No. 6,461,840entitled “Terephthalic acid producing proteobacteria” and U.S. Pat. No.6,187,569 entitled “Microbial production of terephthalic acid andisophthalic acid.”

In certain embodiments, the pH sufficient to maintain the p-toluate orterephthalate in soluble form is between about 5.0-9.0, between about5.5-9.0, between about 6.0-9.0, between about 6.5-9.0, between about7.0-9.0, between about 5.5-8.0, between about 6.0-8.0, or between about6.5-8.0 pH units. In certain embodiments, the pH sufficient to maintainthe p-toluate or terephthalate in soluble form is about 5.5, about 6.0,about 6.5, about 7.0, about 7.5, about 8.0, or about 8.5 pH units. Incertain embodiments, the pH sufficient to maintain the p-toluate orterephthalate in soluble form is about 7.0 pH units. In certainembodiments, the pH sufficient to maintain p-toluate or terephthalate insoluble form is a neutral pH, for example a pH of about 7.0 typicallybounded on its lower end by pH of about 5.0, 5.5 or 6.0 and on its upperend by a pH of about 9.0, 8.5 or 8.0.

The pH of the culture medium can be maintained at a desired pH, whichcan, for example, be a pH between about 5.0-9.0, by addition of a baseor acid, as needed to maintain the culture medium at the desired pH.

Those of skill in the art will understand that the dianionic salts ofterephthalic acid are soluble at concentrations >100 g/L (up to 13% byweight) at 25° C. In certain embodiments, a base is added to the culturemedium in sufficient quantities to maintain terephthalic acid in solubleform. In certain embodiments, a base is added to the culture medium insufficient quantities to maintain the culture medium at a pH betweenabout 5.0-9.0.

It will be understood that counterions, including for instance, sodium,potassium, ammonium, among others, can be added to the culture medium toproduce a desired counterion to an aromatic carboxylate anion, to theextent that the form of the counterion, when added, maintains a desiredpH. In certain embodiments, a base, such as sodium hydroxide (NaOH),potassium hydroxide (KOH), sodium bicarbonate (NaHCO₃), or other bases,or acid, are added as needed to maintain the culture medium at adesirable pH.

In certain embodiments, anhydrous ammonia (NH₃) is added as a base tothe culture medium to maintain the pH around 7. In certain embodiments,a solution of ammonia, also known as ammonium hydroxide (NH₄OH), ammoniawater, ammonical liquor, ammonia liquor, aqua ammonia, aqueous ammonia,or ammonia, is added as a base to the culture medium to maintain the pHaround 7. Use of ammonia as a base offers certain advantages forrecovery and recycling of salts in the processes disclosed herein(described herein and in section entitled “Recovery of Salts forRecycling”).

Those skilled in the art will understand that the aromatic carboxylateanions produced by the biosynthetic routes described herein, i.e.,p-toluate and terephthalate, will be in the form of soluble carboxylatesalts, i.e., the carboxylate anions will be solvated in solution with acounter ion (a cation) in the culture medium. The choice of base used tomaintain the culture medium at a neutral pH will determine the counterion and the type of salt. For example, where a sodium base, such as NaOHor NaHCO₃ is used to maintain neutral pH, the counter ion will be sodium(Na⁺), and a soluble sodium carboxylate salt will be formed. Similarly,where KOH is used, the counter ion will be potassium (K⁺) and a solublepotassium carboxylate salt will be formed. Where ammonia is used, thecounter ion will be ammonium (NH₄ ⁺), and a soluble ammonium carboxylatesalt will be formed. Where the aromatic carboxylic anion is a diacid,for example, terephthalate, a disalt (e.g., disodium, dipotassium, ordiammonium terephthalate) will be formed.

In certain embodiments, following fermentation, the culture medium canbe separated from the cells and/or any non-soluble material by, forexample, centrifugation, filtration, or technique used in the art, toprovide a cell-free medium or broth comprising the aromatic carboxylateanion, prior to the contacting step (described herein and in sectionentitled “Acidification of the Culture Medium to Precipitate theAromatic Carboxylic Acid”). Exemplary filtrations can be micro, nano orultra filtration. Centrifugation and filtration methods are well knownto those skilled in the art.

Acidification of the Culture Medium to Precipitate the AromaticCarboxylic Acid

In certain embodiments, the aromatic carboxylate anion produced by thebiosynthetic routes described herein can be isolated by acidification ofthe culture medium. For example, in certain embodiments, a process isprovided comprising lowering the pH of the culture medium to produce anaromatic carboxylic acid precipitate.

It will be understood that the expressions “acidifying” or“acidification” as used herein mean the addition of substance, acid orotherwise, to an aqueous solution, for example, a culture medium, toresult in that solution having a lower pH. Thus, for instance, it willbe recognized that contacting a culture medium with CO₂ is anacidification of the culture medium.

In certain embodiments, the pH of the culture medium comprising thearomatic carboxylate anion is lowered to less than about 5.0 pH units,less than about 4.5 pH units, less than about 4.0 pH units, less thanabout 3.5 pH units, less than about 3.0 pH units, less than about 2.5 pHunits, less than about 2.0 pH units, less than about 1.5 pH units, orless than about 1.0 pH units. In certain embodiments, the pH of theculture medium is lowered to less than about 3.0 pH units. In certainembodiments, the pH of the culture medium is lowered to less than about2.0 pH units.

Those skilled in the art will understand that the pH of the culturemedium is optimally lowered to a pH value less than the pKa value of thearomatic carboxylic acid to be isolated.

In certain embodiments, the aqueous solubility of the aromaticcarboxylate anions and/or salts disclosed herein is greater than about100 g/L at room temperature. In certain embodiments, the aqueoussolubility of the aromatic carboxylic acids disclosed herein is lessthan 1 g/L at room temperature.

In certain embodiments, acidification of the culture medium results inprecipitation of the aromatic carboxylic acid. In certain embodiments, adecrease in pH of the culture medium results in precipitation of thearomatic carboxylic acid.

In certain embodiments, the precipitate is comprised of a monoacid, forexample, p-toluic acid. In certain embodiments, the precipitate iscomprised of a diacid, for example, terephthalic acid.

In certain embodiments, acidification results in the culture mediumbeing substantially depleted of the aromatic carboxylate anion. Incertain embodiments, a decrease in pH of the culture medium results inthe culture medium being substantially depleted of the aromaticcarboxylate anion. In certain embodiments, precipitation of the aromaticcarboxylic acid results in the culture medium being substantiallydepleted of the aromatic carboxylate anion.

As used herein, the term “substantially depleted” is understood to meanthat less than about 50% of the total amount of aromatic carboxylateanion produced biosynthetically remains in the culture medium followingacidification. In certain embodiments, less than about 50%, less thanabout 40%, less than about 30%, less than about 20%, less than about10%, less than about 5%, or less than about 1% of the aromaticcarboxylate anion remains in the culture medium following acidification.In certain embodiments, “substantially depleted” means that the amountof aromatic carboxylate anion in the culture medium is less than thatwhich can measurably detected.

In certain embodiments, the pH of the culture medium is lowered bycontacting the culture medium with an acid. Such acids can, for example,be those known in the art, including, for instance, hydrochloric,phosphoric, sulfuric, and so forth. In certain embodiments, the acid iscarbon dioxide. It is believed use of carbon dioxide is advantageous inthat, it can, for example, be obtained as a product from the culturedmicrobial organism and/or easily recovered and recycled from theacidified culture medium.

In certain embodiments, the pH of the culture medium is lowered throughcontact with sufficient carbon dioxide (CO₂) to lower the pH of theculture medium to produce the precipitate comprising the aromaticcarboxylic acid.

Those skilled in the art will understand that when carbon dioxidedissolves in water it exists in chemical equilibrium producing carbonicacid (H₂CO₃):

CO₂+H₂O

H₂CO₂,

which in sufficient quantities can be used to lower the pH of theculture medium.

In certain embodiments, the culture medium is contacted with a solutionof CO₂ in water, for example, a solution of carbonic acid (H₂CO₃). Incertain embodiments, the culture medium is contacted with gaseous CO₂.In certain embodiments, the gaseous CO₂ is pure CO₂ gas. In certainembodiments, the gaseous CO₂ is in a mixture with one or more additionalgases. In certain embodiments, the one or more additional gases includenitrogen, an inert gas (helium, argon, etc.), or the like.

In certain embodiments, the culture medium is stirred while it iscontacted with gaseous CO₂.

In certain embodiments, the culture medium is contacted with gaseous CO₂for up to 20 hours.

To provide sufficient H₂CO₃ in solution in the culture medium, thetemperature and pressure of the culture medium can be adjusted. Thoseskilled in the art will understand that the optimum temperature andpressure will depend on the pKa of the aromatic carboxylic acid to beprecipitated, the concentration of the aromatic carboxylate anion, andthe fraction of aromatic carboxylate anion required to be converted tothe aromatic carboxylic acid.

In certain embodiments, the culture medium is pressurized in the rangeof about 1 to 300 atm with CO₂. In certain embodiments, the culturemedium is pressurized in the range of about 50 to 200 atm with CO₂. Incertain embodiments, the culture medium is pressurized in the range ofabout 1 to 100 atm with CO₂. In certain embodiments, the culture mediumis pressurized in the range of about 20 to 50 atm with CO₂. In certainembodiments, the culture medium is pressurized in the range of about 10to 50 atm with CO₂.

More particularly, lower atmospheres of CO₂, between, for example, 1 to30 atm with CO₂ may be useful. In certain embodiments, the culturemedium is pressurized in the range of about 1 to 30 atm with CO₂. Incertain embodiments, the culture medium is contacted with CO₂ underpressure in the range of about 1 to about 20 atm, about 2 to about 25atm, about 5 to about 20 atm, about 10 to about 20 atm, about 13 toabout 20 atm, about 14 to about 20 atm, about 15 to about 20 atm, about14 to about 16 atm, about 16 to about 20 atm, about 17 to about 25 atm,about 2 to about 20 atm, or about 10 to about 16 atm. In certainembodiments, the culture medium is contacted with CO₂ under about 0.01,0.1, 0.5, 1.0, 2, 5, 10, 14, 15, 16, or 20 atm with CO₂.

Where the culture media is contacted with “sufficient” CO₂, by“sufficient,” it is meant that the amount CO₂ lowers the pH of theculture medium to result in an aromatic carboxylic acid precipitate tosubstantially deplete the culture medium of the aromatic carboxyateanion. In certain embodiments, “sufficient” CO₂ comprises contacting theculture media with CO₂ under a pressure and/or temperatures as describedin the paragraphs above and below.

In certain embodiments, the temperature of the culture medium is in therange of about 0° C. to 90° C. In certain embodiments, the temperatureof the culture medium is in the range of about 0° C. to 80° C. Incertain embodiments, the temperature of the culture medium is in therange of about 0° C. to 60° C. In certain embodiments, the temperatureof the culture medium is in the range of about 5° C. to 50° C. Incertain embodiments, the temperature of the culture medium is in therange of about 5° C. to 10° C., 10° C. to 20° C., 20° C. to 30° C., 30°C. to 40° C., 40° C. to 50° C., or 40° C. to 60° C. In certainembodiments, the temperature of the culture medium is about 5, 10, 15,20, 25, 30, 35, 40, 45 or 50° C.

In certain embodiments, the contacting step takes place in a vessel. Insuch embodiments, the pressures and temperatures, and ranges thereof,provided above are those inside the vessel.

In certain embodiments, the vessel is pressurized in the range of about0.1 to 30 atm with CO₂ and the pressurized culture medium in the vesselis stirred at temperatures between about 0° C. and about 80° C. for upto 24 hours.

Trapping and Recycling Side Product from Culturing Step

In certain embodiments, a side product of the culturing step is trappedand recycled. In certain embodiments, the side product of the culturingstep is CO₂. For example, the biosynthetic pathways to 2H3M4OP,exemplified in FIGS. 1-3, each generate one equivalent of CO₂. Carbondioxide may also be produced in other metabolic pathways when culturingthe non-naturally occurring microbial organism. The production of thisCO₂ if not collected and reused represents inefficiency and, ifreleased, has a potential to contribute to Green House Gas emissions.Production of CO₂ by the culture medium is considered bio-based CO₂ byvirtue of the fact that is has the expected ¹⁴C content measured by, forexample, ASTM D6866-05 analysis methods for dating radioactive carbon.

In certain embodiments, a process as provided herein further comprisescollecting CO₂ produced by culturing the non-naturally occurringorganism. The collected CO₂ can, for example, be used to lower the pH ofthe culture medium to produce the aromatic carboxylic acid precipitate,or, for example, to supplement the CO₂ used to lower the pH of theculture medium.

Accordingly, one objective is to improve overall carbon capture and/orimprove overall costs and efficiencies of the processes disclosed hereinfor isolating an aromatic carboxylic acid.

In certain embodiments, CO₂ generated in the biosynthesis of thearomatic carboxylic acid can be trapped and recycled for later use,using, for example, a fermentation trap or CO₂ capture device.

In certain embodiments, CO₂ generated in the biosynthesis of thearomatic carboxylic acid can be trapped and recycled for use to lowerthe pH of the culture medium to precipitate the aromatic carboxylicacid.

In certain embodiments, CO₂ generated, for example, in culturingorganisms in the biosynthesis of the aromatic carboxylic acid can betrapped and recycled for use to maintain anaerobic or substantiallyanaerobic conditions in the culture medium.

Technologies for collecting (also termed trapping, sequestering,recovering) for CO₂ are known including those applicable to collectingCO₂ from culturing organisms. It will understood that such methods can,for example, be employed in connection with collecting CO₂ from thecultured organisms as described herein.

In certain embodiments, CO₂ generated in the biosynthesis of thearomatic carboxylic acid can be trapped and recycled for use as a sourceof carbon to the non-naturally occurring microorganism in the culturemedium. In certain embodiments, CO₂ can be trapped and mixed withSynthesis gas, also known as syngas or producer gas, for use as a sourceof carbon to the non-naturally occurring microorganism in the culturemedium, as disclosed in U.S. Patent Publication No. US 2011/0207185 A1.Syngas is a mixture primarily of H₂ and CO and can be obtained from thegasification of any organic feedstock, including but not limited tocoal, coal oil, natural gas, biomass, and waste organic matter. Althoughlargely H₂ and CO, syngas can also include CO₂ and other gases insmaller quantities. Thus, synthesis gas provides a cost effective sourceof gaseous carbon such as CO and, additionally, CO₂.

Recovery of Salts for Recycling

The aqueous solution remaining after the aromatic carboxylic acid isprecipitated from the culture medium contains solvated salts as counterions, which can be recovered for recycling in the processes disclosedherein. For example, where CO₂ is used to lower the pH of the culturemedium, the aqueous solution will contain solvated carbonate salts.Where ammonia (NH₃) is used as a base to maintain the aromaticcarboxylate anion in soluble form, the aqueous solution will containsolvated ammonium salts. Where both NH₃ and CO₂ are used, the aqueoussolution will contain solvated ammonium carbonate ((NH₄)₂CO₃).

In certain advantageous embodiments, it is contemplated that solvated(NH₄)₂CO₃ salt remaining in the aqueous solution following precipitationof the aromatic carboxylic acid can be recovered and recycled. Forexample, heating the aqueous solution will decompose the (NH₄)₂CO₃ intoNH₃ and CO₂, as depicted below.

(NH₄)₂CO₃→2NH₃+CO₂+H₂O

The resultant gaseous NH₃ and CO₂ can be separated, recovered and/orrecycled using, for example, a fermentation trap or gas capture device.Recovery/recycling of gaseous NH₃ and CO₂ will reduce the costsassociated with use of these materials and will also reduceenvironmental discharges, for example, Green House Gas emissions in thecase of the CO₂ gas. This will further improve overall carbon captureand/or improve overall costs and efficiencies of the processes disclosedherein for isolating an aromatic carboxylic acid.

In certain embodiments, the NH₃ recovered from decomposition of(NH₄)₂CO₃ can be recycled for use as a base to maintain the aromaticcarboxylate anion in soluble form in the culture medium.

In certain embodiments, the CO₂ recovered from decomposition of(NH₄)₂CO₃ can be recycled for use to lower the pH of the culture mediumto precipitate the aromatic carboxylic acid.

In certain embodiments, the CO₂ recovered from decomposition of(NH₄)₂CO₃ can be recycled for use to maintain anaerobic or substantiallyanaerobic conditions in the culture medium used to produce the aromaticcarboxylate anion.

In certain embodiments, the CO₂ recovered from decomposition of(NH₄)₂CO₃ can be recycled for use as a source of carbon to thenon-naturally occurring microorganism in the culture medium.

The remaining aqueous solution will be at a higher pH, and may also berecycled for use in, for example, the culture medium. In certainembodiments, enough CO₂ will be removed so that the aqueous solutionreturns to a neutral pH to provide a neutral solution for the culturemedium.

In certain embodiments, following acidification and precipitation of thearomatic carboxylic acid, the aqueous solution can be filtered toprovide an aqueous filtrate from which the solvated salts may berecovered for recycling.

Separating the Aromatic Carboxylic Acid from the Culture Medium

Once precipitated, the aromatic carboxylic acid may be separated fromthe other components in the culture medium by removing the culturemedium. In certain embodiments, the separation of the aromaticcarboxylic acid from the other components in the culture mediumcomprises filtering and recovering of the aromatic carboxylic acid fromthe culture medium. In certain embodiments the precipitated aromaticcarboxylic acid is resolubilized and subjected to extraction orseparation procedures well know in the art, which can, for example,include continuous liquid-liquid extraction, pervaporation, membranefiltration, membrane separation, reverse osmosis, electrodialysis,distillation, crystallization, centrifugation, extractive filtration,ion exchange chromatography, size exclusion chromatography, adsorptionchromatography, and ultrafiltration. All of the above methods are wellknown in the art.

In certain embodiments, the aromatic carboxylic acid is purifiedfollowing separation from the other components in the culture medium. Incertain embodiments, the aromatic carboxylic acid is purified usingcrystallization.

Therefore, provided herein is an isolated bio-based p-toluic acidproduced by the processes described herein. Also provided herein is anisolated bio-based terephthalic acid produced by the processes describedherein.

Exemplary Calculations Dependence of Terephthalic Acid Forms on pH

The following describes exemplary calculations of dependence ofterephthalic acid forms on pH.

TABLE 1 Dependence of terephthalic acid forms on pH Total acidUndissociated acid Monoanionic acid Dianioninc acid pH (mol/L) (mol/L)Percent (mol/L) Percent (mol/L) Percent 2 0.354314 0.344349 97.2%0.009931 2.8% 3.44E−05 0.0% 3 0.00421  0.003243 77.0% 0.000935 22.2%3.24E−05 0.8% 4   1E−04 2.05E−05 20.5% 5.91E−05 59.1% 2.05E−05 20.5% 55.67E−06 4.37E−08 0.8% 1.26E−06 22.2% 4.37E−06 77.0% 6 5.02E−07 4.88E−110.0% 1.41E−08 2.8% 4.88E−07 97.2% 6.5 1.43E−07 1.42E−12 0.0% 1.29E−090.9% 1.42E−07 99.1%

Table 1 demonstrates that at a pH value of 4, terephthalic acid formsexists in a ratio of about 20.5/59/20.5 anion to monoacid to diacid; ata pH value of 3, terephthalic acid forms exists in a ratio of about1/22/77 anion to monoacid to diacid; at a pH value of 2, terephthalicacid forms exists in a ratio of about 0/3/97 anion to monoacid todiacid, as illustrated below.

  terephthalate

  terephthalic acid (monoacid form)

  terephthalic acid (diacid form) pH Ratios 2 0 3 97 3 1 22 77 4 20.5 5920.5

Table 1 further demonstrates that at pH 7, close to 100% of terephthalicacid will be in the anionic form. Thus, in the processes providedherein, where the non-naturally occurring microbial organism producesterephthalate, performing the culturing step at a neutral pH willproduce the anion, that is, close to 100% of terephthalic acid will bein the anionic form (terephthalate).

Similarly, a culture medium that is acidified to less than about 3.0 pHunits will afford predominantly the diacid form of terephthalic acid. Itwill be understood that terephthalic acid is virtually insoluble inaqueous solutions (0.017 g/L solubility in water at 25° C.). In lightthat terephthalic acid is not soluble in aqueous solution, it is quiteclear from Table 1 that acidification of the culture medium to valuesless than about 3.0 pH units will result in a substantial depletion ofterephthalate in the culture medium, because at values less than about3.0 pH units, there will no soluble form of terephthalic acid remainingin the culture medium, no matter what the form (anion, monoacid ordiacid).

Pressure-pH Relationship for CO₂ in Water

The following describes exemplary calculations of pressure-pHrelationship for CO₂ in water.

TABLE 2 Pressure-pH relationship for CO₂ in water. [CO₂] [H₂CO₃] [HCO₃⁻] [CO₃ ²⁻] (atm) pH (mol/L) (mol/L) (mol/L) (mol/L) 1.0 × 10⁻⁸ 7.003.36 × 10⁻¹⁰ 5.71 × 10⁻¹³ 1.42 × 10⁻⁰⁹ 7.90 × 10⁻¹³ 1.0 × 10⁻⁷ 6.94 3.36× 10⁻⁰⁹ 5.71 × 10⁻¹² 5.90 × 10⁻⁰⁹ 1.90 × 10⁻¹² 1.0 × 10⁻⁶ 6.81 3.36 ×10⁻⁰⁸ 5.71 × 10⁻¹¹ 9.16 × 10⁻⁰⁸ 3.30 × 10⁻¹¹ 1.0 × 10⁻⁵ 6.42 3.36 ×10⁻⁰⁷ 5.71 × 10⁻⁰⁹ 3.78 × 10⁻⁰⁷ 4.63 × 10⁻¹¹ 1.0 × 10⁻⁴ 5.92 3.36 ×10⁻⁰⁶ 5.71 × 10⁻⁰⁹ 1.19 × 10⁻⁰⁶ 5.67 × 10⁻¹¹ 3.5 × 10⁻⁴ 5.65 1.18 ×10⁻⁰⁵ 2.00 × 10⁻⁰⁸ 2.23 × 10⁻⁰⁶ 6.60 × 10⁻¹¹ 1.0 × 10⁻³ 5.42 3.36 ×10⁻⁰⁵ 5.71 × 10⁻⁰⁸ 3.78 × 10⁻⁰⁶ 5.61 × 10⁻¹¹ 1.0 × 10⁻² 4.92 3.36 ×10⁻⁰⁴ 5.71 × 10⁻⁰⁷ 1.19 × 10⁻⁰⁵ 5.61 × 10⁻¹¹ 1.0 × 10⁻¹ 4.42 3.36 ×10⁻⁰³ 5.71 × 10⁻⁰⁶ 3.78 × 10⁻⁰⁵ 5.61 × 10⁻¹¹ 1.0 × 10⁺⁰ 3.92 3.36 ×10⁻⁰² 5.71 × 10⁻⁰⁵ 1.20 × 10⁻⁰⁴ 5.61 × 10⁻¹¹ 2.5 × 10⁺⁰ 3.72 8.40 ×10⁻⁰² 1.43 × 10⁻⁰⁴ 1.89 × 10⁻⁰⁴ 5.61 × 10⁻¹¹ 1.0 × 10⁺¹ 3.42 3.36 ×10⁻⁰¹ 5.71 × 10⁻⁰⁴ 3.78 × 10⁻⁰⁴ 5.61 × 10⁻¹¹

As demonstrated in Table 2, CO₂ pressures between 1-10 atmospherescorrespond to pH values as low as 3.42. Thus, acidification of aqueousculture media with CO₂ pressures greater than 2.5 atmospheres can, forexample, reduce pH values to less than 4.0.

It is understood that modifications which do not substantially affectthe activity of the various embodiments of this invention are alsoprovided within the definition of the invention provided herein.Accordingly, the following examples are intended to illustrate but notlimit the present invention.

EXAMPLES Example I Exemplary Pathways for Producing 2H3M4OP

This example describes an exemplary pathway for producing theterephthalic acid (PTA) precursor 2H3M4OP.

The precursor to the p-toluate and PTA pathways is 2H3M4OP. FIG. 1 showsa pathway from erythrose-4-phosphate to 2H3M4OP. The first two steps ofthe pathway employ enzymes of P5C biosynthesis. In the first step,oxidation of erythrose-4-phosphate to 4-phosphoerythronate is catalyzedby erythrose-4-phosphate dehydrogenase (EC 1.2.1.72). In step B,4-phosphoerythronate is further oxidized to2-oxo-3-hydroxy-4-phosphobutanoate by 4-phosphoerythronate dehydrogenase(EC 1.1.1.290). The next three steps of the pathway are analogous to theisoleucine biosynthesis pathway comprising a synthase, a ketol-acidreductoisomerase and a diol dehydratase. In step C, a2-acetyl-2,3-dihydroxy-4-phosphobutanoate synthase converts pyruvate and2-oxo-3-hydroxy-4-phosphobutanoate to2-acetyl-2,3-dihydroxy-4-phosphobutanoate, releasing CO₂. Thisintermediate is then converted to2,3,4-trihydroxy-3-methyl-5-phosphopentanoate by a reductoisomerase.Dehydration by a diol dehydratase forms the 2H3M4OP precursor,4-hydroxy-3-methyl-2-oxo-5-phosphopentanoate. 2H3M4OP is then formed bydecarboxylation of the keto-acid.

Pathways from 4,5-dihydroxy-2-oxopentanoate to 2H3M4OP are depicted inFIG. 2. 4,5-dihydroxy-2-oxopentanoate is derived from sugars such asarabinose and xylose. It can also be formed enzymatically bycondensation of pyruvate and glycolaldehyde by aldolase enzymes such as2-dehydro-3-deoxypentonate aldolase, 2-dehydro-3-deoxyglucaratealdolase, or other enzymes in EC class 4.1.2 or 4.1.3. In one 2H3M4OPpathway, 4,5-dihydroxy-2-oxopentanoate is converted to4,5-dihydroxy-3-methyl-2-oxopentanoate by a methyltransferase (step A).The methylated product is then phosphorylated and decarboxylated (stepsB/C). In an alternate pathway, the 4,5-dihydroxy-2-oxopentanoatesubstrate is phosphorylated to 4-hydroxy-2-oxo-5-phosphopentanoate (stepD), then methylated by a methyltransferase (step E) to the4-hydroxy-3-methyl-2-oxo-5-phosphopentanoate intermediate. Both pathwaysshare the final decarboxylation of4-hydroxy-3-methyl-2-oxo-5-phosphopentanoate to 2H3M4OP.

Enzyme Candidates

EC Class Description Pathway step 1.1.1.a Oxidoreductase (alcohol tooxo) 1B 1.1.1.b Ketol-acid reductoisomerase 1D 1.2.1.a Oxidoreductase(aldehyde to acid) 1A 2.2.1.a Synthase 1C 2.7.1.a Kinase 2B, 2D 4.1.1.aDecarboxylase 1F, 2C 4.2.1.b Diol dehydratase 1E No EC Methyltransferase2A, 2E

EC 1.1.1.a

The NAD(P)+ dependent oxidation of erythronate-4-phosphate to2-oxo-3-hydroxy-4-phosphobutanoate is catalyzed by 4-phosphoerythronatedehydrogenase (Step 1B, EC 1.1.1.290). This enzyme, encoded by the pdxBgene of E. coli, participates in pyridoxine biosynthesis (Zhao et al, JBacteriol 177:2804-12 (1995)). An analogous enzyme is encoded by pdxB ofPseudomonas aeruginosa (Ha et. al, J Mol Biol 366:1294-1304 (2007)). TheE. coli and P. aeruginosa enzymes utilize NAD+ as a cofactor. The pdxRgene from Sinorhizobium melitoti utlizes FAD+ as a cofactor (Hoshino etal, WO/2004/029250). Alcohol dehydrogenases that convert 2-hydroxyacidsto 2-ketoacids, such as malate dehydrogenase (mdh) and lactatedehydrogenase (ldhA) of E. coli, are also suitable candidates. Thelactate dehydrogenase from Ralstonia eutropha has been shown todemonstrate high activities on 2-ketoacids of various chain lengthsincludings lactate, 2-oxobutyrate, 2-oxopentanoate and 2-oxoglutarate(Steinbuchel et al., Eur. J. Biochem. 130:329-334 (1983)).

Gene Accession No. GI No. Organism pdxB NP_416823.1 16130255 Escherichiacoli pdxB Q9I3W9.1 46396520 Pseudomonas aeruginosa pdxR AEH77803.1336031871 Sinorhizobium melitoti mdh AAC76268.1 1789632 Escherichia colildhA NP_415898.1 16129341 Escherichia coli ldh YP_725182.1 113866693Ralstonia eutropha

EC 1.1.1.b

The reduction and isomerization of2-acetyl-2,3-dihydroxy-4-phosphobutanoate to2,3,4-trihydroxy-3-methyl-5-phosphopentanoate is catalyzed by abifunctional enzyme with 2-acetyl-2,3-dihydroxy-4-phosphobutanoatereductoisomerase activity (Step 1D). An analogous transformation iscatalyzed by ketol-acid reductoisomerase (EC 1.1.1.86), an enzymeinvolved in branched chain amino acid biosynthesis. This enzyme, encodedby ilvC of Escherichia coli, is active on multiple substrates including2-hydroxy-2-methyl-3-oxobutanoate, 2,3-dihydroxy-3-methylpentanoate and2,3-dihydroxy-3-isopentanoate. Crystal structure of enzymes from E. coliand Pseudomonas aeruginosa are available (Tyagi et al., Prot Sci14:3089-3100 (2005); Ahn et al., J Mol Biol 328:505-15 (2003)).Additional candidates are ilvC of C. glutamicum (Lewal et al, JBiotechnol 104:241-52 (2003)) and ilv5p of Saccharomyces cerevisiae(Omura, Appl Microbiol Biotechnol 78:503-13 (2008)).

Gene Accession No. GI No. Organism ilvC NP_418222.1 16131632 Escherichiacoli ilvC YP_793157.1 116052840 Pseudomonas aeruginosa ilvC EHE83601.1354510679 Corynebacterium glutamicum Ilv5p NP_013459.1 6323387Saccharomyces cerevisiae

EC 1.2.1.a

The oxidation of erythrose-4-phosphate to 4-phosphoerythronate iscatalyzed by an oxidoreductase that converts an aldehyde to an acid. Anenzyme with this activity is erythrose-4-phosphate dehydrogenase (EC1.2.1.72). An NAD+ dependent erythrose-4-dehydrogenase is encoded by theepd gene of E. coli (Yang et al, J Bacteriol 180:4294-99 (1998)). Asimilar enzyme has been characterized in Vibrio cholera (Carroll et al,J Bacteriol 179:293-6 (1997)). Additional candidates are NAD+-dependentaldehyde dehydrogenases (EC 1.2.1.−) and NAD(P)+ dependentglyceraldehyde-3-phosphate dehydrogenases (EC 1.2.1.3 and 1.2.1.9). Twoaldehyde dehydrogenases found in human liver, ALDH-1 and ALDH-2, havebroad substrate ranges for a variety of aliphatic, aromatic andpolycyclic aldehydes (Klyosov, Biochemistry 35:4457-4467 (1996a)).Active ALDH-2 has been efficiently expressed in E. coli using the GroELproteins as chaperonins (Lee et al., Biochem. Biophys. Res. Commun.298:216-224 (2002)). The rat mitochondrial aldehyde dehydrogenase alsohas a broad substrate range (Siew et al., Arch. Biochem. Biophys.176:638-649 (1976)). The NAD+ dependent gapN gene of Thermoproteus tenaxoxidizes glyceraldehyde-3-phosphate to its corresponding acid (Brunneret al, J Biol Chem 273:6149-56 (1998)). An exemplary NADP+ dependentglyceraldehyde-3-phosphate dehydrogenase is the GAPN gene product ofArabidopsis thaliana (Rius et al Plant Mol Biol 61:945-57 (2006))

Gene Accession No. GI No. Organism epd NP_417402.1 16130828 Escherichiacoli epd YP_001216003.2 229259769 Vibrio cholera ALDH-2 P05091.2 118504Homo sapiens ALDH-2 NP_115792.1 14192933 Rattus norvegicus gapNCAA71651.1 3059159 Thermoproteus tenax GAPN ABB83822.1 82570696Arabidopsis thaliana

EC 2.2.1.a

Formation of 2-acetyl-2,3-dihydroxy-4-phosphobutanoate in Step 1C iscatalyzed by a synthase enzyme in EC class 2.2.1. A suitable enzyme forcatalyzing this transformation is acetohydroxyacid synthase (EC2.2.1.6), which condenses pyruvate and 2-oxobutanoate to2-aceto-2-hydroxybutanoate and carbon dioxide. Alternately, twopyruvates are condensed forming acetolactate. The enzyme operates inbranched chain amino acid biosynthesis pathways. Two isozymes are activein E. coli: ilvBN and ilvHI (Vyazmensky et al Biochem, 35:10339-46(1996); Engel et al, Biotechnol bioeng 88:825-31 (2004)). The IlvBNenzyme has activity on a broad range of aldehyde substrates in vitro.The Methanococcus aeolicus ilvBN gene product is similar to the E. coliIlvBN enzyme (Xing et al, J Bacteriol 176:1207-13 (1994)). Theacetolactate synthase from Bacillus subtilis (AlsS), which naturallycatalyzes the condensation of two molecules of pyruvate to form2-acetolactate, is also able to catalyze the decarboxylation of2-ketoisovalerate like KDC both in vivo and in vitro (Atsumi and Liao,AEM 75:6306-11 (2009)).

Gene Accession No. GI No. Organism ilvB NP_418127.1 16131541 Escherichiacoli ilvN NP_418126.1 16131540 Escherichia coli ilvH NP_414620.116128071 Escherichia coli ilvI YP_025294.2 90111084 Escherichia coliilvB AAB53488.1 2065479 Methanococcus aeolicus ilvN AAB53489.1 2065480Methanococcus aeolicus alsS Q04789.3 239938889 Bacillus subtilis

EC 2.7.1.a

Kinases catalyze the ATP-dependent transfer of a phosphate group to analcohol. Kinase enzymes are required to catalyze the phosphorylation of4,5-dihydroxy-3-methyl-2-oxopentanoate (Step 2B) and4,5-dihydroxy-2-oxopentanoate (Step 2D). The enzymes described belownaturally possess such activity or can be engineered to exhibit thisactivity. Kinases that catalyze the transfer of a phosphate group to analcohol group are members of the EC 2.7.1 enzyme class. The table belowlists several useful kinase enzymes in the EC 2.7.1 enzyme class.

Enzyme Commission Number Enzyme Name 2.7.1.1 hexokinase 2.7.1.2glucokinase 2.7.1.3 ketohexokinase 2.7.1.4 fructokinase 2.7.1.5rhamnulokinase 2.7.1.6 galactokinase 2.7.1.7 mannokinase 2.7.1.8glucosamine kinase 2.7.1.10 phosphoglucokinase 2.7.1.116-phosphofructokinase 2.7.1.12 gluconokinase 2.7.1.13dehydrogluconokinase 2.7.1.14 sedoheptulokinase 2.7.1.15 ribokinase2.7.1.16 ribulokinase 2.7.1.17 xylulokinase 2.7.1.18 phosphoribokinase2.7.1.19 phosphoribulokinase 2.7.1.20 adenosine kinase 2.7.1.21thymidine kinase 2.7.1.22 ribosylnicotinamide kinase 2.7.1.23 NAD+kinase 2.7.1.24 dephospho-CoA kinase 2.7.1.25 adenylyl-sulfate kinase2.7.1.26 riboflavin kinase 2.7.1.27 erythritol kinase 2.7.1.28triokinase 2.7.1.29 glycerone kinase 2.7.1.30 glycerol kinase 2.7.1.31glycerate kinase 2.7.1.32 choline kinase 2.7.1.33 pantothenate kinase2.7.1.34 pantetheine kinase 2.7.1.35 pyridoxal kinase 2.7.1.36mevalonate kinase 2.7.1.39 homoserine kinase 2.7.1.40 pyruvate kinase2.7.1.41 glucose-1-phosphate phosphodismutase 2.7.1.42 riboflavinphosphotransferase 2.7.1.43 glucuronokinase 2.7.1.44 galacturonokinase2.7.1.45 2-dehydro-3-deoxygluconokinase 2.7.1.46 L-arabinokinase2.7.1.47 D-ribulokinase 2.7.1.48 uridine kinase 2.7.1.49hydroxymethylpyrimidine kinase 2.7.1.50 hydroxyethylthiazole kinase2.7.1.51 L-fuculokinase 2.7.1.52 fucokinase 2.7.1.53 L-xylulokinase2.7.1.54 D-arabinokinase 2.7.1.55 allose kinase 2.7.1.561-phosphofructokinase 2.7.1.58 2-dehydro-3-deoxygalactonokinase 2.7.1.59N-acetylglucosamine kinase 2.7.1.60 N-acylmannosamine kinase 2.7.1.61acyl-phosphate-hexose phosphotransferase 2.7.1.62 phosphoramidate-hexosephosphotransferase 2.7.1.63 polyphosphate-glucose phosphotransferase2.7.1.64 inositol 3-kinase 2.7.1.65 scyllo-inosamine 4-kinase 2.7.1.66undecaprenol kinase 2.7.1.67 1-phosphatidylinositol 4-kinase 2.7.1.681-phosphatidylinositol-4-phosphate 5-kinase 2.7.1.69protein-Np-phosphohistidine-sugar phosphotransferase 2.7.1.70 identicalto EC 2.7.1.37. 2.7.1.71 shikimate kinase 2.7.1.72 streptomycin 6-kinase2.7.1.73 inosine kinase 2.7.1.74 deoxycytidine kinase 2.7.1.76deoxyadenosine kinase 2.7.1.77 nucleoside phosphotransferase 2.7.1.78polynucleotide 5′-hydroxyl-kinase 2.7.1.79 diphosphate-glycerolphosphotransferase 2.7.1.80 diphosphate-serine phosphotransferase2.7.1.81 hydroxylysine kinase 2.7.1.82 ethanolamine kinase 2.7.1.83pseudouridine kinase 2.7.1.84 alkylglycerone kinase 2.7.1.85 β-glucosidekinase 2.7.1.86 NADH kinase 2.7.1.87 streptomycin 3″-kinase 2.7.1.88dihydrostreptomycin-6-phosphate 3′a-kinase 2.7.1.89 thiamine kinase2.7.1.90 diphosphate-fructose-6-phosphate 1-phosphotransferase 2.7.1.91sphinganine kinase 2.7.1.92 5-dehydro-2-deoxygluconokinase 2.7.1.93alkylglycerol kinase 2.7.1.94 acylglycerol kinase 2.7.1.95 kanamycinkinase 2.7.1.100 S-methyl-5-thioribose kinase 2.7.1.101 tagatose kinase2.7.1.102 hamamelose kinase 2.7.1.103 viomycin kinase 2.7.1.1056-phosphofructo-2-kinase 2.7.1.106 glucose-1,6-bisphosphate synthase2.7.1.107 diacylglycerol kinase 2.7.1.108 dolichol kinase 2.7.1.113deoxyguanosine kinase 2.7.1.114 AMP-thymidine kinase 2.7.1.118ADP-thymidine kinase 2.7.1.119 hygromycin-B 7″-O-kinase 2.7.1.121phosphoenolpyruvate-glycerone phosphotransferase 2.7.1.122 xylitolkinase 2.7.1.127 inositol-trisphosphate 3-kinase 2.7.1.130tetraacyldisaccharide 4′-kinase 2.7.1.134 inositol-tetrakisphosphate1-kinase 2.7.1.136 macrolide 2′-kinase 2.7.1.137 phosphatidylinositol3-kinase 2.7.1.138 ceramide kinase 2.7.1.140 inositol-tetrakisphosphate5-kinase 2.7.1.142 glycerol-3-phosphate-glucose phosphotransferase2.7.1.143 diphosphate-purine nucleoside kinase 2.7.1.144tagatose-6-phosphate kinase 2.7.1.145 deoxynucleoside kinase 2.7.1.146ADP-dependent phosphofructokinase 2.7.1.147 ADP-dependent glucokinase2.7.1.148 4-(cytidine 5′-diphospho)-2-C-methyl-D-erythritol kinase2.7.1.149 1-phosphatidylinositol-5-phosphate 4-kinase 2.7.1.1501-phosphatidylinositol-3-phosphate 5-kinase 2.7.1.151inositol-polyphosphate multikinase 2.7.1.153phosphatidylinositol-4,5-bisphosphate 3-kinase 2.7.1.154phosphatidylinositol-4-phosphate 3-kinase 2.7.1.156 adenosylcobinamidekinase 2.7.1.157 N-acetylgalactosamine kinase 2.7.1.158inositol-pentakisphosphate 2-kinase 2.7.1.159inositol-1,3,4-trisphosphate 5/6-kinase 2.7.1.160 2′-phosphotransferase2.7.1.161 CTP-dependent riboflavin kinase 2.7.1.162 N-acetylhexosamine1-kinase 2.7.1.163 hygromycin B 4-O-kinase 2.7.1.164O-phosphoseryl-tRNASec kinase

Particularly useful kinase enzymes for catalyzing steps 2B and 2D aremevalonate kinase, glycerol kinase, homoserine kinase, glycerate kinaseand erythritol kinase. A good candidate for this step is mevalonatekinase (EC 2.7.1.36) that phosphorylates the terminal hydroxyl group ofmevalonate. Some gene candidates for this step are erg12 from S.cerevisiae, mvk from Methanocaldococcus jannaschi, MVK from Homosapiens, and mvk from Arabidopsis thaliana col.

Protein GenBank ID GI Number Organism erg12 CAA39359.1 3684Saccharomyces cerevisiae mvk Q58487.1 2497517 Methanocaldococcusjannaschii mvk AAH16140.1 16359371 Homo sapiens mvk NP_851084.1 30690651Arabidopsis thaliana

Glycerol kinase also phosphorylates the terminal hydroxyl group inglycerol to form glycerol-3-phosphate. This reaction occurs in severalspecies, including Escherichia coli, Saccharomyces cerevisiae, andThermotoga maritima. The E. coli glycerol kinase has been shown toaccept alternate substrates such as dihydroxyacetone and glyceraldehyde(Hayashi et al., J Biol. Chem. 242:1030-1035 (1967)). T, maritime hastwo glycerol kinases (Nelson et al., Nature 399:323-329 (1999)).Glycerol kinases have been shown to have a wide range of substratespecificity. Crans and Whiteside studied glycerol kinases from fourdifferent organisms (Escherichia coli, S. cerevisiae, Bacillusstearothermophilus, and Candida mycoderma) (Crans et al., J. Am. Chem.Soc. 107:7008-7018 (2010); Nelson et al., Nature 399:323-329 (1999)).They studied 66 different analogs of glycerol and concluded that theenzyme could accept a range of substituents in place of one terminalhydroxyl group and that the hydrogen atom at C2 could be replaced by amethyl group. Interestingly, the kinetic constants of the enzyme fromall four organisms were very similar.

Protein GenBank ID GI Number Organism glpK AP_003883.1 89110103Escherichia coli glpK1 NP_228760.1 15642775 Thermotoga maritime glpK2NP_229230.1 15642775 Thermotoga maritime Gut1 NP_011831.1 82795252Saccharomyces cerevisiae

Homoserine kinase is another candidate kinase. This enzyme is present ina number of organisms including E. coli, Streptomyces sp, and S.cerevisiae. Homoserine kinase from E. coli has been shown to haveactivity on numerous substrates, including, L-2-amino,1,4-butanediol,aspartate semialdehyde, and 2-amino-5-hydroxyvalerate (Huo et al.,Biochemistry 35:16180-16185 (1996); Huo et al., Arch. Biochem. Biophys.330:373-379 (1996)). This enzyme can act on substrates where thecarboxyl group at the alpha position has been replaced by an ester or bya hydroxymethyl group.

Protein GenBank ID GI Number Organism thrB BAB96580.2 85674277Escherichia coli SACT1DRAFT_4809 ZP_06280784.1 282871792 Streptomycessp. ACT-1 Thr1 AAA35154.1 172978 Saccharomyces serevisiae

The interconversion of 3-phosphoglycerate and glycerate is catalyzed byglycerate kinase (EC 2.7.1.31). Three classes of glycerate kinase havebeen identified. Enzymes in class I and II produceglycerate-2-phosphate, whereas the class III enzymes found in plants andyeast produce glycerate-3-phosphate (Bartsch et al., FEBS Lett.582:3025-3028 (2008)). In a recent study, class III glycerate kinaseenzymes from Saccharomyces cerevisiae, Oryza sativa and Arabidopsisthaliana were heterologously expressed in E. coli and characterized(Bartsch et al., FEBS Lett. 582:3025-3028 (2008)).

Protein GenBank ID GI Number Organism glxK AAC73616.1 1786724Escherichia coli YGR205W AAS56599.1 45270436 Saccharomyces cerevisiaeOs01g0682500 BAF05800.1 113533417 Oryza sativa At1g80380 BAH57057.1227204411 Arabidopsis thaliana

Erythritol is converted to erythritol-4-phosphate by the erythritolkinase. Erythritol kinase (EC 2.7.1.27) catalyzes the phosphorylation oferythritol. Erythritol kinase was characterized in erythritol utilizingbacteria such as Brucella abortus (Sperry et al., J Bacteriol.121:619-630 (1975)). The eryA gene of Brucella abortus has beenfunctionally expressed in Escherichia coli and the resultant EryA wasshown to catalyze the ATP-dependent conversion of erythritol toerythritol-4-phosphate (Lillo et al., Bioorg. Med. Chem. Lett.13:737-739 (2003)).

Protein GenBank ID GI Number Organism eryA Q8YCU8 81850596 Brucellamelitensis eriA Q92NH0 81774560 Sinorhizobium meliloti eryAYP_001108625.1 134102964 Saccharopolyspora erythraea NRRL 2338

EC 4.1.1.a

Decarboxylation of 4-hydroxy-3-methyl-2-oxo-5-phosphopentanoate to2H3M4OP (Steps 1F and 2C) is catalyzed by a keto-acid decarboxylase.Although an enzyme with 2H3M4OP-forming activity has not been describedin the literature, the decarboxylation of keto-acids is catalyzed by avariety of enzymes with varied substrate specificities, includingpyruvate decarboxylase (EC 4.1.1.1), benzoylformate decarboxylase (EC4.1.1.7), alpha-ketoglutarate decarboxylase and branched-chainalpha-ketoacid decarboxylase, phosphonopyruvate decarboxylase,sulfopyruvate decarboxylase, acetohydroxy acid synthase/acetolactatesynthase, glyoxylate carboligase and indole pyruvate decarboxylase.

Branched chain alpha-ketoacid decarboxylase (BCKAD, EC 4.1.1.72) is aparticularly useful enzyme for the invention, as the pathway substrateis also a branched alpha-ketoacid. This class of enzyme has been shownto decarboxylate a variety of compounds varying in chain length from 3to 6 carbons (Oku et al., J Biol Chem. 263:18386-18396 (1988); Smit etal., Appl Environ Microbiol 71:303-311 (2005)). The BCKAD enzyme inLactococcus lactis has been characterized on a variety of branched andlinear substrates including 2-oxobutanoate, 2-oxohexanoate,2-oxopentanoate, 3-methyl-2-oxobutanoate, 4-methyl-2-oxobutanoate andisocaproate (Smit et al., Appl Environ Microbiol 71:303-311 (2005)). Theenzyme has been structurally characterized (Berg et al., Science.318:1782-1786 (2007)). Sequence alignments between the Lactococcuslactis enzyme and the pyruvate decarboxylase of Zymomonas mobilusindicate that the catalytic and substrate recognition residues arenearly identical (Siegert et al., Protein Eng Des Sel 18:345-357(2005)), so this enzyme would be a promising candidate for directedengineering of substrate specificity. Several ketoacid decarboxylases ofSaccharomyces cerevisiae catalyze the decarboxylation of branchedsubstrates, including ARO10, PDC6, PDC5, PDC1 and THI3 (Dickenson et al,J Biol Chem 275:10937-42 (2000)). Yet another BCKAD enzyme is encoded byrv0853c of Mycobacterium tuberculosis (Werther et al, J Biol Chem283:5344-54 (2008)). This enzyme is subject to allosteric activation byalpha-ketoacid substrates. Decarboxylation of alpha-ketoglutarate by aBCKA was detected in Bacillus subtilis; however, this activity was low(5%) relative to activity on other branched-chain substrates (Oku andKaneda, J Biol Chem. 263:18386-18396 (1988)) and the gene encoding thisenzyme has not been identified to date. Additional BCKA gene candidatescan be identified by homology to the Lactococcus lactis proteinsequence. Many of the high-scoring BLASTp hits to this enzyme areannotated as indolepyruvate decarboxylases (EC 4.1.1.74). Indolepyruvatedecarboxylase (IPDA) is an enzyme that catalyzes the decarboxylation ofindolepyruvate to indoleacetaldehyde in plants and plant bacteria.Recombinant branched chain alpha-keto acid decarboxylase enzymes derivedfrom the E1 subunits of the mitochondrial branched-chain keto aciddehydrogenase complex from Homo sapiens and Bos taurus have been clonedand functionally expressed in E. coli (Davie et al., J. Biol. Chem.267:16601-16606 (1992); Wynn et al., J. Biol. Chem. 267:12400-12403(1992); Wynn et al., J. Biol. Chem. 267:1881-1887 (1992)). In thesestudies, the authors found that co-expression of chaperonins GroEL andGroES enhanced the specific activity of the decarboxylase by 500-fold(Wynn et al., J. Biol. Chem. 267:12400-12403 (1992)).

Protein GenBank ID GI Number Organism kdcA AAS49166.1 44921617Lactococcus lactis PDC6 NP_010366.1 6320286 Saccharomyces cerevisiaePDC5 NP_013235.1 6323163 Saccharomyces cerevisiae PDC1 P06169 30923172Saccharomyces cerevisiae ARO10 NP_010668.1 6320588 Saccharomycescerevisiae THI3 NP_010203.1 6320123 Saccharomyces cerevisiae rv0853cO53865.1 81343167 Mycobacterium tuberculosis BCKDHB NP_898871.1 34101272Homo sapiens BCKDHA NP_000700.1 11386135 Homo sapiens BCKDHB P21839115502434 Bos taurus BCKDHA P11178 129030 Bos taurus

Another class of enzymes suitable for decarboxylating a phosphorylatedalpha-ketoacid such as 4-hydroxy-3-methyl-2-oxo-5-phosphopentanoate isphosphonopyruvate decarboxylase (EC 4.1.1.82). This enzyme catalyzes thedecarboxylation of 3-phosphonopyruvate to 2-phosphonoacetaldehyde.Exemplary phosphonopyruvate decarboxylase enzymes are encoded by dhpF ofStreptomyces luridus, ppd of Streptomyces viridochromogenes, fom2 ofStreptomyces wedmorensis (Circello et al, Chem Biol 17:402-11 (2010);Blodgett et al, FEMS Microbiol Lett 163:149-57 (2005); Hidaka et al, MolGen Genet 249:274-80 (1995)). The Bacteroides fragilis enzyme, encodedby aepY, also decarboxylates pyruvate and sulfopyruvate (Zhang et al, JBiol Chem 278:41302-8 (2003)).

Protein GenBank ID GI Number Organism dhpF ACZ13457.1 268628095Streptomyces luridus Ppd CAJ14045.1 68697716 Streptomycesviridochromogenes Fom2 BAA32496.1 1061008 Streptomyces wedmorensis aepYAAG26466.1 11023509 Bacteroides fragilis

Other useful ketoacid decarboxylases include pyruvate decarboxylaseenzymes such as the pdc gene product of Zymomonas mobilis. This enzymehas a broad substrate range and has been a subject of directedengineering studies to alter the affinity for different substrates(Siegert et al., Protein Eng Des Sel 18:345-357 (2005)). The crystalstructure of this enzyme is available (Killenberg-Jabs et al., Eur. J.Biochem. 268:1698-1704 (2001)). The benzoylformate decarboxylase fromPseudomonas putida has a broad substrate range and has been the targetof enzyme engineering studies (Polovnikova et al., 42:1820-1830 (2003);Hasson et al., 37:9918-9930 (1998)). Site-directed mutagenesis of tworesidues in the active site of the Pseudomonas putida enzyme altered theaffinity (Km) of naturally and non-naturally occurring substrates(Siegert et al., Protein Eng Des Sel 18:345-357 (2005)). The propertiesof this enzyme have been further modified by directed engineering(Lingen et al., Chembiochem. 4:721-726 (2003); Lingen et al., ProteinEng 15:585-593 (2002)). The benzoylformate decarboxylase fromPseudomonas aeruginosa, encoded by mdlC, has also been characterizedexperimentally (Barrowman et al., 34:57-60 (1986)). Additional genecandidates from Pseudomonas stutzeri, Pseudomonas fluorescens and otherorganisms can be inferred by sequence homology or identified using agrowth selection system developed in Pseudomonas putida (Henning et al.,Appl. Environ. Microbiol. 72:7510-7517 (2006)). Alpha-ketoglutaratedecarboxylases are also relevant to the invention. An exemplary KDC isencoded by kad in Mycobacterium tuberculosis (Tian et al., PNAS102:10670-10675 (2005)). KDC enzyme activity has also been detected inseveral species of rhizobia including Bradyrhizobium japonicum andMesorhizobium loti (Green et al., J Bacteriol 182:2838-2844 (2000)). Anovel class of AKG decarboxylase enzymes has recently been identified incyanobacteria such as Synechococcus sp. PCC 7002 and homologs (Zhang andBryant, Science 334:1551-3 (2011)).

Protein GenBank ID GI Number Organism pdc P06672.1 118391 Zymomonasmobilis mdlC P20906.2 3915757 Pseudomonas putida mdlC Q9HUR2.1 81539678Pseudomonas aeruginosa dpgB ABN80423.1 126202187 Pseudomonas stutzeriilvB-1 YP_260581.1 70730840 Pseudomonas fluorescens kgd O50463.4160395583 Mycobacterium tuberculosis kgd NP_767092.1 27375563Bradyrhizobium japonicum USDA110 kgd NP_105204.1 13473636 Mesorhizobiumloti ilvB ACB00744.1 169887030 Synechococcus sp. PCC 7002

EC 4.2.1.a

Formation of 4-hydroxy-3-methyl-2-oxo-5-phosphopentanoate from2,3,4-trihydroxy-3-methyl-5-phosphopentanoate (Step 1E) is catalyzed bya diol dehydratase enzyme in EC class 4.2.1. Exemplary diol dehydrataseenzymes are listed in the table below.

Enzyme Commission Number Enzyme Name 4.2.1.5 arabinonate dehydratase4.2.1.6 galactonate dehydratase 4.2.1.7 altronate dehydratase 4.2.1.8mannonate dehydratase 4.2.1.9 dihydroxy-acid dehydratase 4.2.1.12phosphogluconate dehydratase 4.2.1.25 L-arabinonate dehydratase 4.2.1.28propanediol dehydratase 4.2.1.30 glycerol dehydratase 4.2.1.32L(+)-tartrate dehydratase 4.2.1.39 gluconate dehydratase 4.2.1.40glucarate dehydratase 4.2.1.41 5-dehydro-4-deoxyglucarate dehydratase4.2.1.42 galactarate dehydratase 4.2.1.43 2-dehydro-3-deoxy-L-arabinonate dehydratase 4.2.1.44 myo-inosose-2 dehydratase 4.2.1.45CDP-glucose 4,6-dehydratase 4.2.1.46 dTDP-glucose 4,6-dehydratase4.2.1.47 GDP-mannose 4,6-dehydratase 4.2.1.76 UDP-glucose4,6-dehydratase 4.2.1.81 D(−)-tartrate dehydratase 4.2.1.82 xylonatedehydratase 4.2.1.90 L-rhamnonate dehydratase 4.2.1.109methylthioribulose 1- phosphate dehydratase

Particularly useful diol dehydratase enzymes include dihydroxy-aciddehydratase (EC 4.2.1.9), phosphogluconate dehydratase (EC 4.2.1.12) andarabonate dehydratase (EC 4.2.1.25). Dihydroxy-acid dehydratase (DHAD,EC 4.2.1.9) is a B12-independent enzyme participating in branched-chainamino acid biosynthesis. In its native role, it converts2,3-dihydroxy-3-methylvalerate to 2-keto-3-methyl-valerate, a precursorof isoleucine. In valine biosynthesis the enzyme catalyzes thedehydration of 2,3-dihydroxy-isovalerate to 2-oxoisovalerate. The DHADfrom Sulfolobus solfataricus has a broad substrate range and activity ofa recombinant enzyme expressed in E. coli was demonstrated on a varietyof aldonic acids (KIM et al., J. Biochem. 139:591-596 (2006)). The S.solfataricus enzyme is tolerant of oxygen unlike many diol dehydrataseenzymes. The E. coli enzyme, encoded by ilvD, is sensitive to oxygen,which inactivates its iron-sulfur cluster (Flint et al., J. Biol. Chem.268:14732-14742 (1993)). Similar enzymes have been characterized inNeurospora crassa (Altmiller et al., Arch. Biochem. Biophys. 138:160-170(1970)), Salmonella typhimurium (Armstrong et al., Biochim. Biophys.Acta 498:282-293 (1977)) and Corynebacterium glutamicum (Holatko et al,J Biotechnol 139:203-10 (2009)). Other groups have shown that theoverexpression of one or more Aft proteins or homologs thereof improvesDHAD activity (US Patent Application 2011/0183393. In Saccharomycescerevisiae, the Aft1 and Aft2 proteins are transcriptional activatorsthat regulate numerous proteins related to the acquisition,compartmentalization, and utilization of iron. Phosphogluconatedehydratase is another keto-acid forming diol dehydratase that is activeon a phosphorylated substrate. This enzyme participates in the pentosephosphate pathway catalyzing the conversion of 6-phosphogluconate to2-dehydro-3-deoxygluconate-6-phosphate. These enzymes areB12-independent and contain an Fe—S cluster in the active site.Exemplary enzymes are the edd gene products of E. coli (Egan et al, JBacteriol 174:4638-46 (1992)), Pseudomonas aeruginosa (Cuskey et al, JBacteriol 162:865-71 (1985)) and Xanthomonas oryzae (Kim et al,Biotechnol Lett 32:527-31 (2010)). Another suitable enzyme is arabonatedehydratase. A gene encoding this activity is araC of Azospirillumbrasilense (Watanabe et al, J Biol Chem 287:33521-36 (2006)). Additionaldiol dehydratase candidates are described in Example II.

Protein GenBank ID GI Number Organism ilvD NP_344419.1 15899814Sulfolobus solfataricus ilvD AAT48208.1 48994964 Escherichia coli ilvDNP_462795.1 16767180 Salmonella typhimurium ilvD XP_958280.1 85090149Neurospora crassa ilvD CAB57218.1 6010023 Corynebacterium glutamicumAft1 P22149.2 1168370 Saccharomyces cerevisiae Aft2 Q08957.1 74583775Saccharomyces cerevisiae edd NP_416365.1 16129804 Escherichia coli eddNP_251884.1 15598390 Pseudomonas aeruginosa edd YP_001913412.1 188576483Xanthomonas oryzae araC BAE94269.1 95102048 Azospirillum brasilense

No EC

Step 2A depicts 4,5-dihydroxy-2-oxopentanoate methyltransferase whichcatalyzes the methylation of 4.5-dihydroxy-2-oxopentanoate to form4,5-dihydroxy-3-methyl-2-oxopentanoate. A similar transformation iscatalyzed by 4-hydroxy-2-oxo-5-phosphopentanoate methyltransferase inStep 2E. Although enzymes with these activities have not been identifiedto date, methyltransferase enzymes that react with similar substratesinclude alpha-ketoglutarate methyltransferase and keto-argininemethyltransferase. Alpha-ketoglutarate methyltransferase is catalyzed bythe products of genes glmT from Streptomyces coelicolor, dptl fromStreptomyces roseosporus, and lptI from Streptomyces fradiae (Mahlert etal., J. Am. Chem. Soc., 2007, 129 (39), 12011-12018). Keto-argininemethyltransferase is encoded by mrsA of Pseudomonas syringae (Braun etal, AEM 76:2500-8 (2010)).

Protein GenBank ID GI Number Organism glmT NP_627429.1 21221650Streptomyces coelicolor dptI ZP_04706744.1 239986080 Streptomycesroseosporus lptI AAZ23087.1 71068232 Streptomyces fradiae mrsAACY54549.1 262342060 Pseudomonas syringae

Example II Exemplary Pathway for Producing 2H3M4OP fromGlyceraldehyde-3-phosphate (G3P) and Pyruvate

This example describes an exemplary pathway for producing theterephthalic acid (PTA) precursor 2H3M4OP.

The precursor to the p-toluate and PTA pathways is 2H3M4OP. Thischemical can be derived from central metabolitesglyceraldehyde-3-phosphate (G3P) and pyruvate in three enzymatic stepsas shown in FIG. 3. The first two steps are native to E. coli and otherorganisms that utilize the methylerythritol phosphate (non-mevalonate)pathway for isoprenoid biosynthesis. Pyruvate and G3P are firstcondensed to form 1-deoxy-D-xylulose 5-phosphate (DXP) by DXP synthase.Subsequent reduction and rearrangement of the carbon backbone iscatalyzed by DXP reductoisomerase. Finally, a novel diol dehydratasetransforms 2-C-methyl-D-erythritol-4-phosphate to the p-toluateprecursor 2H3M4OP.

A. 1-Deoxyxylulose-5-phosphate (DXP) synthase

Pyruvate and G3P are condensed to form DXP by DXP synthase (EC 2.2.1.7).This enzyme catalyzes the first step in the non-mevalonate pathway ofisoprenoid biosynthesis. The enzyme requires thiamine diphosphate as acofactor, and also requires reduced FAD, although there is no net redoxchange. A crystal structure of the E. coli enzyme is available (Xiang etal., J. Biol. Chem. 282:2676-2682 (2007)). Other enzymes have beencloned and characterized in M. tuberculosis (Bailey et al., Glycobiology12:813-820 (2002) and Agrobacterium tumefaciens (Lee et al., J.Biotechnol. 128:555-566 (2007). DXP synthase enzymes from B. subtilisand Synechocystis sp. PCC 6803 were cloned into E. coli (Harker andBramley, FEBS Lett. 448:115-119 (1999).

GenBank Gene Accession No. GI No. Organism dxs AAC73523.1 1786622Escherichia coli dxs P0A554.1 61222979 M. tuberculosis dxs11 AAP56243.137903541 Agrobacterium tumefaciens dxs P54523.1 1731052 Bacillussubtilis sll1945 BAA17089.1 1652165 Synechocystis sp. PCC 6803

B. 1-Deoxy-D-xylulose-5-phosphate reductoisomerase (EC 1.1.1.267)

The NAD(P)H-dependent reduction and rearrangement of1-deoxy-D-xylulose-5-phosphate (DXP) to2-C-methyl-D-erythritol-4-phosphate is catalyzed by DXP reductoisomerase(DXR, EC 1.1.1.267) in the second step of the non-mevalonate pathway forisoprenoid biosynthesis. The NADPH-dependent E. coli enzyme is encodedby dxr (Takahashi et al., Proc. Natl. Acad. Sci. USA 95:9879-9884(1998)). A recombinant enzyme from Arabidopsis thaliana was functionallyexpressed in E. coli (Carretero-Paulet et al., Plant Physiol.129:1581-1591 (2002). DXR enzymes from Zymomonas mobilis andMycobacterium tuberculosis have been characterized and crystalstructures are available (Grolle et al., FEMS Microbiol. Lett.191:131-137 (2000); Henriksson et al., Acta Crystallogr. D. Biol.Crystallogr. 62:807-813 (2006). Most characterized DXR enzymes arestrictly NADPH dependent, but the enzymes from A. thaliana and M.tuberculosis react with NADH at a reduced rate (Argyrou and Blanchard,Biochemistry 43:4375-4384 (2004); Rohdich et al., FEBS J. 273:4446-4458(2006)).

GenBank Accession Gene No. GI No. Organism dxr AAC73284.1 1786369Escherichia coli dxr AAF73140.1 8131928 Arabisopsis thaliana dxrCAB60758.1 6434139 Zymomonas mobilis dxr NP_217386.2 57117032Mycobacterium tuberculosis

C. 2-C-Methyl-D-erythritol-4-phosphate dehydratase

A diol dehydratase is required to convert2-C-methyl-D-erythritol-4-phosphate into the p-toluate precursor(Altmiller and Wagner, Arch. Biochem. Biophys. 138:160-170 (1970)).Although this transformation has not been demonstrated experimentally,several enzymes catalyze similar transformations includingdihydroxy-acid dehydratase (EC 4.2.1.9), propanediol dehydratase (EC4.2.1.28), glycerol dehydratase (EC 4.2.1.30), myo-inositose dehydratase(EC 4.2.1.44), 2-keto-3-deoxyarabonate dehydratase (EC 4.2.1.43),phosphogluconate dehydratase (EC 4.2.1.12) and arabonate dehydratase.These enzymes are described in further detail below. Additional dioldehydratase enzyme candidates are described above in Example 1.

Diol dehydratase or propanediol dehydratase enzymes (EC 4.2.1.28)capable of converting the secondary diol 2,3-butanediol to 2-butanoneare excellent candidates for this transformation.Adenosylcobalamin-dependent diol dehydratases contain alpha, beta andgamma subunits, which are all required for enzyme function. Exemplarygene candidates are found in Klebsiella pneumoniae (Tobimatsu et al.,Biosci. Biotechnol. Biochem. 62:1774-1777 (1998); Toraya et al.,Biochem. Biophys. Res. Commun. 69:475-480 (1976)), Salmonellatyphimurium (Bobik et al., J. Bacteriol. 179:6633-6639 (1997)),Klebsiella oxytoca (Tobimatsu et al., J. Biol. Chem. 270:7142-7148(1995)) and Lactobacillus collinoides (Sauvageot et al., FEMS Microbiol.Lett. 209:69-74 (2002)). Methods for isolating diol dehydratase genecandidates in other organisms are well known in the art (see, forexample, U.S. Pat. No. 5,686,276).

GenBank Accession Gene No. GI No. Organism pddA BAA08099.1 868006Klebsiella oxytoca pddB BAA08100.1 868007 Klebsiella oxytoca pddCBAA08101.1 868008 Klebsiella oxytoca pduC AAB84102.1 2587029 Salmonellatyphimurium pduD AAB84103.1 2587030 Salmonella typhimurium pduEAAB84104.1 2587031 Salmonella typhimurium pduC CAC82541.1 18857678Lactobacullus collinoides pduD CAC82542.1 18857679 Lactobaculluscollinoides pduE CAD01091.1 18857680 Lactobacullus collinoides pddAAAC98384.1 4063702 Klebsiella pneumoniae pddB AAC98385.1 4063703Klebsiella pneumoniae pddC AAC98386.1 4063704 Klebsiella pneumoniae

Enzymes in the glycerol dehydratase family (EC 4.2.1.30) can also beused to dehydrate 2-C-methyl-D-erythritol-4-phosphate. Exemplary genecandidates encoded by gldABC and dhaB123 in Klebsiella pneumoniae (WO2008/137403) and (Toraya et al., Biochem. Biophys. Res. Commun.69:475-480 (1976)), dhaBCE in Clostridium pasteuranum (Macis et al.,FEMS Microbiol Lett. 164:21-28 (1998)) and dhaBCE in Citrobacterfreundii (Seyfried et al., J. Bacteriol. 178:5793-5796 (1996)). Variantsof the B12-dependent diol dehydratase from K. pneumoniae with 80- to336-fold enhanced activity were recently engineered by introducingmutations in two residues of the beta subunit (Qi et al., J. Biotechnol.144:43-50 (2009)). Diol dehydratase enzymes with reduced inactivationkinetics were developed by DuPont using error-prone PCR (WO2004/056963).

GenBank Accession Gene No. GI No. Organism gldA AAB96343.1 1778022Klebsiella pneumoniae gldB AAB96344.1 1778023 Klebsiella pneumoniae gldCAAB96345.1 1778024 Klebsiella pneumoniae dhaB1 ABR78884.1 150956854Klebsiella pneumoniae dhaB2 ABR78883.1 150956853 Klebsiella pneumoniaedhaB3 ABR78882.1 150956852 Klebsiella pneumoniae dhaB AAC27922.1 3360389Clostridium pasteuranum dhaC AAC27923.1 3360390 Clostridium pasteuranumdhaE AAC27924.1 3360391 Clostridium pasteuranum dhaB P45514.1 1169287Citrobacter freundii dhaC AAB48851.1 1229154 Citrobacter freundii dhaEAAB48852.1 1229155 Citrobacter freundii

If a B12-dependent diol dehydratase is utilized, heterologous expressionof the corresponding reactivating factor is recommended. B12-dependentdiol dehydratases are subject to mechanism-based suicide activation bysubstrates and some downstream products. Inactivation, caused by a tightassociation with inactive cobalamin, can be partially overcome by dioldehydratase reactivating factors in an ATP-dependent process.Regeneration of the B12 cofactor requires an additional ATP. Dioldehydratase regenerating factors are two-subunit proteins. Exemplarycandidates are found in Klebsiella oxytoca (Mori et al., J. Biol. Chem.272:32034-32041 (1997)), Salmonella typhimurium (Bobik et al., J.Bacteriol. 179:6633-6639 (1997); Chen et al., J. Bacteriol.176:5474-5482 (1994)), Lactobacillus collinoides (Sauvageot et al., FEMSMicrobiol. Lett. 209:69-74 (2002)), and Klebsiella pneumonia (WO2008/137403).

GenBank Accession Gene No. GI No. Organism ddrA AAC15871.1 3115376Klebsiella oxytoca ddrB AAC15872.1 3115377 Klebsiella oxytoca pduGAAL20947.1 16420573 Salmonella typhimurium pduH AAL20948.1 16420574Salmonella typhimurium pduG YP_002236779 206579698 Klebsiella pneumoniapduH YP_002236778 206579863 Klebsiella pneumonia pduG CAD01092 29335724Lactobacillus collinoides pduH CAD01093 29335725 Lactobacilluscollinoides

B12-independent diol dehydratase enzymes utilize S-adenosylmethionine(SAM) as a cofactor, function under strictly anaerobic conditions, andrequire activation by a specific activating enzyme (Frey et al., Chem.Rev. 103:2129-2148 (2003)). The glycerol dehydrogenase and correspondingactivating factor of Clostridium butyricum, encoded by dhaB1 and dhaB2,have been well-characterized (O'Brien et al., Biochemistry 43:4635-4645(2004); Raynaud et al., Proc. Natl. Acad. Sci USA 100:5010-5015 (2003)).This enzyme was recently employed in a 1,3-propanediol overproducingstrain of E. coli and was able to achieve very high titers of product(Tang et al., Appl. Environ. Microbiol. 75:1628-1634 (2009)). Anadditional B12-independent diol dehydratase enzyme and activating factorfrom Roseburia inulinivorans was shown to catalyze the conversion of2,3-butanediol to 2-butanone (US publication 2009/09155870).

GenBank Accession Gene No. GI No. Organism dhaB1 AAM54728.1 27461255Clostridium butyricum dhaB2 AAM54729.1 27461256 Clostridium butyricumrdhtA ABC25539.1 83596382 Roseburia inulinivorans rdhtB ABC25540.183596383 Roseburia inulinivorans

Dihydroxy-acid dehydratase (DHAD, EC 4.2.1.9) is a B12-independentenzyme participating in branched-chain amino acid biosynthesis. In itsnative role, it converts 2,3-dihydroxy-3-methylvalerate to2-keto-3-methyl-valerate, a precursor of isoleucine. In valinebiosynthesis, the enzyme catalyzes the dehydration of2,3-dihydroxy-isovalerate to 2-oxoisovalerate. The DHAD from Sulfolobussolfataricus has a broad substrate range, and activity of a recombinantenzyme expressed in E. coli was demonstrated on a variety of aldonicacids (Kim and Lee, J. Biochem. 139:591-596 (2006)). The S. solfataricusenzyme is tolerant of oxygen unlike many diol dehydratase enzymes. TheE. coli enzyme, encoded by ilvD, is sensitive to oxygen, whichinactivates its iron-sulfur cluster (Flint et al., J. Biol. Chem.268:14732-14742 (1993)). Similar enzymes have been characterized inNeurospora crassa (Altmiller and Wagner, Arch. Biochem. Biophys.138:160-170 (1970)) and Salmonella typhimurium (Armstrong et al.,Biochim. Biophys. Acta 498:282-293 (1977)).

GenBank Accession Gene No. GI No. Organism ilvD NP_344419.1 15899814Sulfolobus solfataricus ilvD AAT48208.1 48994964 Escherichia coli ilvDNP_462795.1 16767180 Salmonella typhimurium ilvD XP_958280.1 85090149Neurospora crassa

The diol dehydratase myo-inosose-2-dehydratase (EC 4.2.1.44) is anotherexemplary candidate. Myo-inosose is a six-membered ring containingadjacent alcohol groups. A purified enzyme encodingmyo-inosose-2-dehydratase functionality has been studied in Klebsiellaaerogenes in the context of myo-inositol degradation (Berman andMagasanik, J. Biol. Chem. 241:800-806 (1966)), but has not beenassociated with a gene to date. The myo-inosose-2-dehydratase ofSinorhizobium fredii was cloned and functionally expressed in E. coli(Yoshida et al., Biosci. Biotechnol. Biochem. 70:2957-2964 (2006)). Asimilar enzyme from B. subtilis, encoded by iolE, has also been studied(Yoshida et al., Microbiology 150:571-580 (2004)).

GenBank Accession Gene No. GI No. Organism iolE P42416.1 1176989Bacillus subtilis iolE AAX24114.1 60549621 Sinorhizobium fredii

2-Keto-3-deoxyarabonate dehydratase participates in arabinosedegradation in Azospirillum brasilense, converting2-keto-3-deoxyarabonate to alpha-ketoglutarate semialdehyde (Watanabe etal, J Biol Chem 287:33521-36 (2006)). Similar enzymes can be identifiedby sequence homology. Phosphogluconate dehydratase is a diol dehydratasethat participates in the pentose phosphate pathway catalyzing theconversion of 6-phosphogluconate to2-dehydro-3-deoxygluconate-6-phosphate. Phosphogluconate dehydrataseenzymes are B12-independent and contain an Fe—S cluster in the activesite. Exemplary enzymes are the edd gene products of E. coli (Egan etal, J Bacteriol 174:4638-46 (1992)), Pseudomonas aeruginosa (Cuskey etal, J Bacteriol 162:865-71 (1985)) and Xanthomonas oryzae (Kim et al,Biotechnol Lett 32:527-31 (2010)). Another suitable enzyme is arabonatedehydratase. A gene encoding this activity is araC of Azospirillumbrasilense (Watanabe et al, J Biol Chem 287:33521-36 (2006)).

Protein GenBank ID GI Number Organism araD BAE94270.1 95102049Azospirillum brasilense dapA ZP_02907581.1 171318425 Burkholderiaambifaria BCAM2800 P_002235400.1 206564637 Burkholderia cenocepacia eddNP_416365.1 16129804 Escherichia coli edd NP_251884.1 15598390Pseudomonas aeruginosa edd YP_001913412.1 188576483 Xanthomonas oryzaearaC BAE94269.1 95102048 Azospirillum brasilense

Example III Exemplary Pathway for Synthesis of p-Toluate from 2H3M4OP byShikimate Pathway Enzymes

This example describes exemplary pathways for synthesis of p-toluateusing shikimate pathway enzymes.

The chemical structure of p-toluate closely resembles p-hydroxybenzoate,a precursor of the electron carrier ubiquinone. 4-Hydroxybenzoate issynthesized from central metabolic precursors by enzymes in theshikimate pathway, found in bacteria, plants and fungi. The shikimatepathway is comprised of seven enzymatic steps that transformD-erythrose-4-phosphate (E4P) and phosphoenolpyruvate (PEP) tochorismate. Pathway enzymes include 2-dehydro-3-deoxyphosphoheptonate(DAHP) synthase, dehydroquinate (DHQ) synthase, DHQ dehydratase,shikimate dehydrogenase, shikimate kinase,5-enolpyruvylshikimate-3-phosphate (EPSP) synthase and chorismatesynthase. In the first step of the pathway, D-erythrose-4-phosphate andphosphoenolpyruvate are joined by DAHP synthase to form3-deoxy-D-arabino-heptulosonate-7-phosphate. This compound is thendephosphorylated, dehydrated and reduced to form shikimate. Shikimate isconverted to chorismate by the actions of three enzymes: shikimatekinase, 3-phosphoshikimate-2-carboxyvinyltransferase and chorismatesynthase. Subsequent conversion of chorismate to 4-hydroxybenzoate iscatalyzed by chorismate lyase.

The synthesis of p-toluate proceeds in an analogous manner as shown inFIG. 4. The pathway originates with PEP and 2H3M4OP, a compoundanalogous to E4P with a methyl group in place of the 3-hydroxyl group ofE4P. The hydroxyl group of E4P does not directly participate in thechemistry of the shikimate pathway reactions, so the methyl-substituted2H3M4OP precursor is expected to react as an alternate substrate.Directed or adaptive evolution can be used to improve preference for2H3M4OP and downstream derivatives as substrates. Such methods arewell-known in the art.

Strain engineering strategies for improving the efficiency of fluxthrough shikimate pathway enzymes are also applicable here. Theavailability of the pathway precursor PEP can be increased by alteringglucose transport systems (Yi et al., Biotechnol. Prog. 19:1450-1459(2003)). 4-Hydroxybenzoate-overproducing strains were engineered toimprove flux through the shikimate pathway by means of overexpression ofa feedback-insensitive isozyme of 3-deoxy-D-arabinoheptulosonicacid-7-phosphate synthase (Barker and Frost, Biotechnol. Bioeng.76:376-390 (2001)). Additionally, expression levels of shikimate pathwayenzymes and chorismate lyase were enhanced. Similar strategies can beemployed in a strain for overproducing p-toluate.

A. 2-Dehydro-3-deoxyphosphoheptonate synthase (EC 2.5.1.54)

The condensation of D-erythrose-4-phosphate and phosphoenolpyruvate iscatalyzed by 2-dehydro-3-deoxyphosphoheptonate (DAHP) synthase (EC2.5.1.54). Three isozymes of this enzyme are encoded in the E. coligenome by aroG, aroF and aroH and are subject to feedback inhibition byphenylalanine, tyrosine and tryptophan, respectively. In wild-type cellsgrown on minimal medium, the aroG, aroF and aroH gene productscontributed 80%, 20% and 1% of DAHP synthase activity, respectively(Hudson and Davidson, J. Mol. Biol. 180:1023-1051 (1984)). Two residuesof AroG were found to relieve inhibition by phenylalanine (Kikuchi etal., Appl. Environ. Microbiol. 63:761-762 (1997)). The feedbackinhibition of AroF by tyrosine was removed by a single base-pair change(Weaver and Herrmann, J. Bacteriol. 172:6581-6584 (1990)). Thetyrosine-insensitive DAHP synthase was overexpressed in a4-hydroxybenzoate-overproducing strain of E. coli (Barker and Frost,Biotechnol. Bioeng. 76:376-390 (2001)). The aroG gene product was shownto accept a variety of alternate 4- and 5-carbon length substrates(Sheflyan et al., J. Am. Chem. Soc. 120(43):11027-11032 (1998);Williamson et al., Bioorg. Med. Chem. Lett. 15:2339-2342 (2005)). Theenzyme reacts efficiently with (3S)-2-deoxyerythrose-4-phosphate, asubstrate analogous to D-erythrose-4-phosphate but lacking the alcoholat the 2-position (Williamson et al., supra 2005). Enzymes fromHelicobacter pylori and Pyrococcus furiosus also accept this alternatesubstrate (Schofield et al., Biochemistry 44:11950-11962 (2005); Webbyet al., Biochem. J. 390:223-230 2005)) and have been expressed in E.coli. An evolved variant of DAHP synthase, differing from the wild typeE. coli AroG enzyme by 7 amino acids, was shown to exhibit a 60-foldimprovement in Kcat/K_(M) (Ran and Frost, J. Am. Chem. Soc.129:6130-6139 (2007)).

GenBank Accession Gene No. GI No. Organism aroG AAC73841.1 1786969Escherichia coli aroF AAC75650.1 1788953 Escherichia coli aroHAAC74774.1 1787996 Escherichia coli aroF Q9ZMU5 81555637 Helicobacterpylori PF1690 NP_579419.1 18978062 Pyrococcus furiosus

B. 3-Dehydroquinate synthase (EC 4.2.3.4)

The dephosphorylation of substrate(2)(2,4-dihydroxy-5-methyl-6-[(phosphonooxy)methyl]oxane-2-carboxylate)to substrate (3)(1,3-dihydroxy-4-methylcylohex-1-ene-1-carboxylate) asshown in FIG. 4 is analogous to the dephosphorylation of3-deoxy-arabino-heptulonate-7-phosphate by 3-dehydroquinate synthase.The enzyme has been characterized in E. coli (Mehdi et al., MethodsEnzymol. 142:306-314 (1987), B. subtilis (Hasan and Nester, J. Biol.Chem. 253:4999-5004 (1978)) and Mycobacterium tuberculosis H37Rv (deMendonca et al., J. Bacteriol. 189:6246-6252 (2007)). The E. coli enzymeis subject to inhibition by L-tyrosine (Barker and Frost, Biotechnol.Bioeng. 76:376-390 2001)).

GenBank Accession Gene No. GI No. Organism aroB AAC76414.1 1789791Escherichia coli aroB NP_390151.1 16079327 Bacillus subtilis aroBCAB06200.1 1781064 Mycobacterium tuberculosis

C. 3-Dehydroquinate dehydratase (EC 4.2.1.10)

3-Dehydroquinate dehydratase, also termed 3-dehydroquinase (DHQase),naturally catalyzes the dehydration of 3-dehydroquinate to3-dehydroshikimate, analogous to step C in the p-toluate pathway of FIG.4. DHQase enzymes can be divided into two classes based on mechanism,stereochemistry and sequence homology (Gourley et al., Nat. Struct.Biol. 6:521-525. (1999)). Generally the type 1 enzymes are involved inbiosynthesis, while the type 2 enzymes operate in the reverse(degradative) direction. Type 1 enzymes from E. coli (Kinghorn et al.,Gene 14:73-80. 1981)), Salmonella typhi (Kinghorn et al., supra 1981;Servos et al., J. Gen. Microbiol. 137:147-152 (1991)) and B. subtilis(Warburg et al., Gene 32:57-66 1984)) have been cloned andcharacterized. Exemplary type II 3-dehydroquinate dehydratase enzymesare found in Mycobacterium tuberculosis, Streptomyces coelicolor (Evanset al., FEBS Lett. 530:24-30 (2002)) and Helicobacter pylori (Lee etal., Proteins 51:616-7 (2003)).

GenBank Accession Gene No. GI No. Organism aroD AAC74763.1 1787984Escherichia coli aroD P24670.2 17433709 Salmonella typhi aroCNP_390189.1 16079365 Bacillus subtilis aroD P0A4Z6.2 61219243Mycobacterium tuberculosis aroQ P15474.3 8039781 Streptomyces coelicoloraroQ Q48255.2 2492957 Helicobacter pylori

D. Shikimate Dehydrogenase (EC 1.1.1.25)

Shikimate dehydrogenase catalyzes the NAD(P)H dependent reduction of3-dehydroshikimate to shikimate, analogous to Step D of FIG. 4. The E.coli genome encodes two shikimate dehydrogenase paralogs with differentcofactor specificities. The enzyme encoded by aroE is NADPH specific,whereas the ydiB gene product is a quinate/shikimate dehydrogenase whichcan utilize NADH (preferred) or NADPH as a cofactor (Michel et al., J.Biol. Chem. 278:19463-19472 (2003). NADPH-dependent enzymes fromMycobacterium tuberculosis (Zhang et al., J. Biochem. Mol. Biol.38:624-631 (2005)), Haemophilus influenzae (Ye et al., J. Bacteriol.185:4144-4151 (2003)) and Helicobacter pylori (Han et al., FEBS J.273:4682-4692 (2006)) have been functionally expressed in E. coli.

GenBank Accession Gene No. GI No. Organism aroE AAC76306.1 1789675Escherichia coli ydiB AAC74762.1 1787983 Escherichia coli aroENP_217068.1 15609689 Mycobacterium tuberculosis aroE P43876.1 1168510Haemophilus influenzae aroE AAW22052.1 56684731 Helicobacter pylori

E. Shikimate Kinase (EC 2.7.1.71)

Shikimate kinase catalyzes the ATP-dependent phosphorylation of the3-hydroxyl group of shikimate analogous to Step E of FIG. 4. Twoshikimate kinase enzymes are encoded by aroK (SK1) and aroL (SK2) in E.coli (DeFeyter and Pittard, J. Bacteriol. 165:331-333 (1986);Lobner-Olesen and Marinus, J. Bacteriol. 174:525-529 (1992)). The Km ofSK2, encoded by aroL, is 100-fold lower than that of SK1, indicatingthat this enzyme is responsible for aromatic biosynthesis (DeFeyter etal., supra 1986). Additional shikimate kinase enzymes from Mycobacteriumtuberculosis (Gu et al., J. Mol. Biol. 319:779-789 (2002)); Oliveira etal., Protein Expr. Purif. 22:430-435 (2001)), Helicobacter pylori (Chenget al., J. Bacteriol. 187:8156-8163 (2005)) and Erwinia chrysanthemi(Krell et al., Protein Sci. 10:1137-1149 (2001)) have been cloned in E.coli.

GenBank Accession Gene No. GI No. Organism aroK YP_026215.2 90111581Escherichia coli aroL NP_414922.1 16128373 Escherichia coli aroKCAB06199.1 1781063 Mycobacterium tuberculosis aroK NP_206956.1 15644786Helicobacter pylori SK CAA32883.1 42966 Erwinia chrysanthemi

F. 3-Phosphoshikimate-2-carboxyvinyltransferase (EC 2.5.1.19)

3-Phosphoshikimate-2-carboxyvinyltransferase, also known as5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), catalyzes thetransfer of the enolpyruvyl moiety of phosphoenolpyruvate to the5-hydroxyl of shikimate-3-phosphate. The enzyme is encoded by aroA in E.coli (Anderson et al., Biochemistry 27:1604-1610 (1988)). EPSPS enzymesfrom Mycobacterium tuberculosis (Oliveira et al., Protein Expr. Purif.22:430-435 (2001)), Dunaliella salina (Yi et al., J. Microbiol.45:153-157 (2007)) and Staphylococcus aureus (Priestman et al., FEBSLett. 579:728-732 (2005)) have been cloned and functionally expressed inE. coli.

GenBank Accession Gene No. GI No. Organism aroA AAC73994.1 1787137Escherichia coli aroA AAA25356.1 149928 Mycobacterium tuberculosis aroAAAA71897.1 152956 Staphylococcus aureus aroA ABM68632.1 122937807Dunaliella salina

G. Chorismate Synthase (EC 4.2.3.5)

Chorismate synthase is the seventh enzyme in the shikimate pathway,catalyzing the transformation of 5-enolpyruvylshikimate-3-phosphate tochorismate. The enzyme requires reduced flavin mononucleotide (FMN) as acofactor, although the net reaction of the enzyme does not involve aredox change. In contrast to the enzyme found in plants and bacteria,the chorismate synthase in fungi is also able to reduce FMN at theexpense of NADPH (Macheroux et al., Planta 207:325-334 (1999)).Representative monofunctional enzymes are encoded by aroC of E. coli(White et al., Biochem. J. 251:313-322 (1988)) and Streptococcuspneumoniae (Maclean and Ali, Structure 11:1499-1511 (2003)).Bifunctional fungal enzymes are found in Neurospora crassa (Kitzing etal., J. Biol. Chem. 276:42658-42666 (2001)) and Saccharomyces cerevisiae(Jones et al., Mol. Microbiol. 5:2143-2152 (1991)).

GenBank Accession Gene No. GI No. Organism aroC NP_416832.1 16130264Escherichia coli aroC ACH47980.1 197205483 Streptococcus pneumoniaeU25818.1:19 . . . AAC49056.1 976375 Neurospora crassa 1317 ARO2CAA42745.1 3387 Saccharomyces cerevisiae

H. Chorismate Lyase (EC 4.1.3.40)

Chorismate lyase catalyzes the first committed step in ubiquinonebiosynthesis: the removal of pyruvate from chorismate to form4-hydroxybenzoate. The enzymatic reaction is rate-limited by the slowrelease of the 4-hydroxybenzoate product (Gallagher et al., Proteins44:304-311 (2001)), which is thought to play a role in delivery of4-hydroxybenzoate to downstream membrane-bound enzymes. The chorismatelyase of E. coli was cloned and characterized and the enzyme has beencrystallized (Gallagher et al., supra 2001; Siebert et al., FEBS Lett.307:347-350 (1992)). Structural studies implicate the G90 residue ascontributing to product inhibition (Smith et al., Arch. Biochem.Biophys. 445:72-80 (2006)). Modification of two surface-active cysteineresidues reduced protein aggregation (Holden et al., Biochim. Biophys.Acta 1594:160-167 (2002)). A recombinant form of the Mycobacteriumtuberculosis chorismate lyase was cloned and characterized in E. coli(Stadthagen et al., J. Biol. Chem. 280:40699-40706 2005)).

GenBank Accession Gene No. GI No. Organism ubiC AAC77009.2 87082361Escherichia coli Rv2949c NP_217465.1 15610086 Mycobacterium tuberculosis

B-F. Multifunctional AROM Protein.

In most bacteria, the enzymes of the shikimate pathway are encoded byseparate polypeptides. In microbial eukaryotes, five enzymatic functionsare catalyzed by a polyfunctional protein encoded by a pentafunctionalsupergene (Campbell et al., Int. J. Parasitol. 34:5-13 (2004)). Themultifunctional AROM protein complex catalyzes reactions analogous toreactions B-F of FIG. 4. The AROM protein complex has been characterizedin fungi including Aspergillus nidulans, Neurospora crassa,Saccharomyces cerevisiae and Pneumocystis carinii (Banerji et al., J.Gen. Microbiol. 139:2901-2914 (1993); Charles et al., Nucleic Acids Res.14:2201-2213 (1986); Coggins et al., Methods Enzymol. 142:325-341(1987); Duncan, K., Biochem. J. 246:375-386 (1987)). Several componentsof AROM have been shown to function independently as individualpolypeptides. For example, dehydroquinate synthase (DHQS) forms theamino-terminal domain of AROM, and can function independently whencloned into E. coli (Moore et al., Biochem. J. 301 (Pt 1):297-304(1994)). Several crystal structures of AROM components from Aspergillusnidulans provide insight into the catalytic mechanism (Carpenter et al.,Nature 394:299-302 (1998)).

GenBank Accession Gene No. GI No. Organism AROM P07547.3 238054389Aspergillus nidulans AROM P08566.1 114166 Saccharomyces cerevisiae AROMP07547.3 238054389 Aspergillus nidulans AROM Q12659.1 2492977Pneumocystis carinii

Example IV Exemplary Pathway for Enzymatic Transformation of p-Toluateto Terephthalic Acid

This example describes exemplary pathways for conversion of p-toluate toterephthalic acid (PTA).

P-toluate can be further transformed to PTA by oxidation of the methylgroup to an acid in three enzymatic steps as shown in FIG. 5. Thepathway is comprised of a p-toluate methyl-monooxygenase reductase, a4-carboxybenzyl alcohol dehydrogenase and a 4-carboxybenzyl aldehydedehydrogenase. In the first step, p-toluate methyl-monooxyngenaseoxidizes p-toluate to 4-carboxybenzyl alcohol in the presence of O₂. TheComamonas testosteroni enzyme (tsaBM), which also reacts with 4-toluenesulfonate as a substrate, has been purified and characterized (Locher etal., J. Bacteriol. 173:3741-3748 (1991)). 4-Carboxybenzyl alcohol issubsequently converted to an aldehyde by 4-carboxybenzyl alcoholdehydrogenase (tsaC). The aldehyde to acid transformation is catalyzedby 4-carboxybenzaldehyde dehydrogenase (tsaD). Enzymes catalyzing thesereactions are found in Comamonas testosteroni T-2, an organism capableof utilizing p-toluate as the sole source of carbon and energy (Junkeret al., J. Bacteriol. 179:919-927 (1997)). Additional genes to transformp-toluate to PTA can be found by sequence homology, in particular toproteobacteria in the genera Burkholderia, Alcaligenes, Pseudomonas,Shingomonas and Comamonas (U.S. Pat. No. 6,187,569 and US publication2003/0170836). Genbank identifiers associated with the Comamonastestosteroni enzymes are listed below.

GenBank Accession Gene No. GI No. Organism tsaB AAC44805.1 1790868Comamonas testosteroni tsaM AAC44804.1 1790867 Comamonas testosteronitsaC AAC44807.1 1790870 Comamonas testosteroni tsaD AAC44808.1 1790871Comamonas testosteroni

Example V Exemplary Process for Production and Recovery of TerephthalicAcid

The following example describes an exemplary process for production andrecovery of terephthalic acid from a culture medium, as exemplified andillustrated in FIG. 6.

Culturing the Non-Naturally Occurring Microbial Organism to ProduceTerephthalate:

Sugars can be fermented in a culture medium at neutral pH using anon-naturally occurring microbial organism containing a functionalterephthalate pathway. Under these conditions, terephthalate thusproduced will be in the culture medium of the cells.

If conventional sugars are employed, cells can be removed bycentrifugation or membrane filtration. If biomass is used as a primaryfeedstock, a pretreated stream of biomass can be introduced into thefermenter along with cellulolytic enzymes in order to engage in asimultaneous saccharification and fermentation process.

Ammonia can be used as a base to maintain pH at 7, in which case thefermentation process will produce diammonium terephthalate, which issoluble in the aqueous culture medium.

Following fermentation, once sufficient quantities of terephthalate areproduced, the culture medium can be separated from any non-solublematerials by centrifugation or membrane filtration to provide acell-free broth containing the terephthalate salt. Where biomass is usedas a primary feedstock, both cells and any non-soluble lignocellulosicmaterial remaining can be removed together through centrifugation ormembrane filtration to provide a cell-free broth containing theterephthalate salt, which is soluble in the cell-free broth.

Lowering the pH with CO₂ to Precipitate the Terephthalic Acid:

The cell-free broth can be acidified by adding gaseous carbon dioxide(CO₂). The vessel can be pressurized in the range of 0.1 to 30 atm withCO₂ and allowed to stir at temperatures between 0° C. and 50° C. for upto 20 hours. Lowering the pH of the cell-free broth leads to theinsoluble terephthalic acid precipitating from solution, where theresulting ammonium carbonate ((NH₄)₂CO₃) remains in solution.

Filtering and Recovering the Terephthalic Acid:

The crude terephthalic acid can be filtered from the solution andrecovered. The remaining aqueous filtrate contains (NH₄)₂CO₃, and thissolution can be heated to decompose (NH₄)₂CO₃ into NH₃ and CO₂ forseparation, recovery, and recycling of both or either of thesematerials. Crude terephthalic acid solid can be recrystallized usingstandard techniques and solvents to afford purified terephthalic acid.

Using this exemplary process, a high predicted fermentation yield of upto 0.58 lb terephthalic acid/lb sugar may be obtained.

Throughout this application various publications have been referenced.The disclosures of these publications in their entireties, includingGenBank and GI number publications, are hereby incorporated by referencein this application in order to more fully describe the state of the artto which this invention pertains. Although the invention has beendescribed with reference to the examples provided above, it should beunderstood that various modifications can be made without departing fromthe spirit of the invention.

1. A non-naturally occurring microbial organism, said microbial organismhaving a (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate (2H3M40P) pathwayand comprising at least one exogenous nucleic acid encoding a 2H3M40Ppathway enzyme expressed in a sufficient amount to produce 2H3M40P,wherein said 2H3M40P pathway comprises a pathway selected from: (1) 1A,IB, 1C, ID, IE and IF; (2) 2A, 2B and 2C; and (3) 2D, 2E and 2C, wherein1 A is an erythrose-4-phosphate dehydrogenase, wherein IB is a4-phosphoerythronate dehydrogenase, wherein 1C is a2-acetyl-2,3-dihydroxy-4-phosphobutanoate synthase, wherein ID is a2-acetyl-2,3-dihydroxy-4-phosphobutanoate reductoisomerase, wherein IEis a 2,3,4-trihydroxy-3-methyl-5-phosphopentanoate dehydratase, whereinIF is a 4-hydroxy-3-methyl-2-oxo-5-phosphopentanoate decarboxylase,wherein 2A is a 4,5-dihydroxy-2-oxopentanoate methyltransferase, wherein2B is a 4,5-dihydroxy-3-methyl-2-oxopentanoate kinase, wherein 2C is a4-hydroxy-3-methyl-2-oxo-5-phosphopentanoate decarboxylase, wherein 2Dis a 4,5-dihydroxy-2-oxopentanoate kinase, wherein 2E is a4-hydroxy-2-oxo-5-phosphopentanoate methyltransferase.
 2. Thenon-naturally occurring microbial organism of claim 1, wherein saidmicrobial organism comprises two, three, four, five or six exogenousnucleic acids each encoding a 2H3M40P pathway enzyme.
 3. Thenon-naturally occurring microbial organism of claim 2, wherein saidmicrobial organism comprises exogenous nucleic acids encoding each ofthe enzymes of at least one of the pathways selected from (1)-(3). 4.The non-naturally occurring microbial organism of any one of claim 1,wherein said exogenous nucleic acid is a heterologous nucleic acid. 5.(canceled)
 6. The non-naturally occurring microbial organism of claim 6,wherein said microbial organism further has a p-toluate pathway andcomprises at least one exogenous nucleic acid encoding a p-toluatepathway enzyme expressed in a sufficient amount to produce p-toluate,wherein said p-toluate pathway comprises 4A, 4B, 4C, 4D, 4E, 4F, 4G and4H, wherein 4A is a 2-dehydro-3-deoxyphosphoheptonate synthase; wherein4B is a 3-dehydroquinate synthase; wherein 4C is a 3-dehydroquinatedehydratase; wherein 4D is a shikimate dehydrogenase; wherein 4E is ashikimate kinase; wherein 4F is a3-phosphoshikimate-2-carboxyvinyltransferase; wherein 4G is a chorismatesynthase and wherein 4H is a chorismate lyase.
 7. The non-naturallyoccurring microbial organism of claim 6, wherein said microbial organismcomprises two, three, four, five, six, seven or eight exogenous nucleicacids each encoding a p-toluate pathway enzyme.
 8. The non-naturallyoccurring microbial organism of claim 7, wherein said microbial organismcomprises exogenous nucleic acids encoding each of the enzymes of saidp-toluate pathway.
 9. The non-naturally occurring microbial organism ofclaim 6, wherein said exogenous nucleic acid is a heterologous nucleicacid.
 10. (canceled)
 11. The non-naturally occurring microbial organismof claim 6, wherein said microbial organism further has a terephthalatepathway and comprises at least one exogenous nucleic acid encoding aterephthalate pathway enzyme expressed in a sufficient amount to produceterephthalate, wherein said terephthalate pathway comprises 5A, 5B and5C, wherein 5A is a p-toluate methyl-monooxygenase reductase, wherein 5Bis a 4-carboxybenzyl alcohol dehydrogenase and wherein 5C is a4-carboxybenzyl aldehyde dehydrogenase.
 12. The non-naturally occurringmicrobial organism of claim 11, wherein said microbial organismcomprises two or three exogenous nucleic acids each encoding aterephthalate pathway enzyme.
 13. The non-naturally occurring microbialorganism of claim 12, wherein said microbial organism comprisesexogenous nucleic acids encoding each of the enzymes of saidterephthalate pathway.
 14. The non-naturally occurring microbialorganism of claim 11, wherein said exogenous nucleic acid is aheterologous nucleic acid.
 15. (canceled)
 16. The non-naturallyoccurring microbial organism of claim 1, wherein said microbial organismis a species of bacteria, yeast or fungus.
 17. A method for producing2H3M40P, comprising culturing the non-naturally occurring microbialorganism of claim 1 under conditions and for a sufficient period of timeto produce 2H3M40P.
 18. A method for producing p-toluate, comprisingculturing the non-naturally occurring microbial organism of claim 6under conditions and for a sufficient period of time to producep-toluate.
 19. A method for producing terephthalate, comprisingculturing the non-naturally occurring microbial organism of claim 11under conditions and for a sufficient period of time to produceterephthalate. 20-22. (canceled)
 23. A composition comprising bio-based2H3M40P and a compound other than said bio-based 2H3M40P, wherein saidcompound is a trace amount of a cellular portion of said microbialorganism of claim 1, bio-based p-toluate and a compound other than saidbio-based p-toluate, wherein said compound is a trace amount of acellular portion of said microbial organism of claim 6, bio-basedterephthalate and a compound other than said bio-based terephthalate,wherein said compound is a trace amount of a cellular portion of saidmicrobial organism of claim 11, culture medium comprising bio-based2H3M40P, wherein said culture medium is separated from said microbialorganism of claim 1, bio-based p-toluate, wherein said culture medium isseparated from said microbial organism of claim 6, or bio-basedterephthalate, wherein said culture medium is separated from saidmicrobial organism of claim
 11. 24-25. (canceled)
 26. A process forisolating a bio-based aromatic carboxylic acid from a culture mediumcomprising: a) culturing a non-naturally occurring microbial organism ina culture medium to produce an aromatic carboxylate anion at a pHsufficient to maintain the aromatic carboxylate anion in soluble form;b) separating the culture medium from non-soluble materials; c)contacting the culture medium with sufficient carbon dioxide (CO₂) tolower the pH of the culture medium to produce an aromatic carboxylicacid precipitate, wherein the culture medium is substantially depletedof the aromatic carboxylate anion; and d) separating the aromaticcarboxylic acid from the culture medium; wherein the aromatic carboxylicacid is p-toluic acid or terephthalic acid. 27-52. (canceled)
 53. Aprocess for obtaining a bio-derived polymer using said bio-basedterephthalate produced by the non-naturally occurring microbial organismof claim
 11. 54. The bio-derived polymer of claim 53, where said polymeris polyethylene terephthalate (PET), polybutyl terephthalate (PBT), orpolytrimethylene terephthalate (PTT). 55-88. (canceled)